Compositions and methods for treating arenavirus infection

ABSTRACT

The invention generally provides compositions and methods of treating or preventing an arenavirus infection, using an agent that inhibits binding of an arenavirus glycoprotein 1 (GP1) polypeptide to transferrin receptor 1 (TfR1). The invention also provides methods of designing or identifying therapeutic agents that bind to or target a GP1 receptor-binding site (RBS) to inhibit arenavirus attachment to a cell, and therapeutic agents identified using the methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/392,729, filed Jun. 8, 2016, the entire contents of which are hereby incorporated by reference herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. CA-13202 and AI-109740 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

In the Western hemisphere, there are at least five arenaviruses that cause human viral hemorrhagic fevers with high case fatality rates. The pathogenic New World arenaviruses include the Junín (JUNV), Machupo (MACV), Guanarito (GTOV), and Sabiá (SBAV) viruses, which, respectively, cause Argentine (AHF), Bolivian, Venezuelan, and “Brazilian” hemorrhagic fever, as well as the most recently described member, Chapare virus (CHPV). Of these, Junín virus (JUNV) is the only hemorrhagic fever virus for which transfusion of JUNV survivor immune plasma containing JUNV neutralizing antibodies (‘passive immunity’) is an established treatment. At present, no treatments exist for MACV, GTOV, and SBAV arenavirus infection, and no therapeutic strategies are available to address the threat of future emerging arenaviruses or arenavirus weaponization.

Accordingly, compositions and methods for treating or preventing arenavirus infection are urgently required, and methods for identifying therapeutic agents for treating or preventing arenavirus infection are urgently needed.

SUMMARY OF THE INVENTION

The invention generally provides compositions and methods of treating or preventing an arenavirus infection, including inhibiting binding of an arenavirus glycoprotein 1 (GP1) polypeptide to transferrin receptor 1 (TfR1) and inhibiting attachment of an arenavirus to a cell. Also provided are methods of designing or identifying therapeutic agents useful for inhibiting binding of an arenavirus glycoprotein 1 (GP1) polypeptide to transferrin receptor 1 (TfR1) by targeting a GP1 receptor-binding site (RBS), and therapeutic agents identified using the methods.

In one aspect, the invention provides a method of inhibiting or preventing binding of a transferrin receptor 1 (TfR1) and a glycoprotein 1 (GP1) of one species of New World arenavirus involving contacting a TfR1 with an antibody or antigen-binding fragment thereof generated by an immune response to a glycoprotein 1 (GP1) of a second species of New World arenavirus.

In another aspect, the invention provides a method of treating or preventing a New World arenavirus infection involving administering to a subject infected or at risk of infection with one species of New World arenavirus an antibody or antigen-binding fragment thereof generated by an immune response to a second species of New World arenavirus.

In another aspect, the invention provides a method of treating or preventing a New World arenavirus infection, involving administering to a subject in need thereof the isolated antibody or antigen-binding fragment thereof according to any aspect delineated herein.

In another aspect, the invention provides a method of inhibiting or preventing binding of a transferrin receptor 1 (TfR1) and an arenavirus glycoprotein 1 (GP1), involving contacting a TfR1 with the isolated antibody or antigen-binding fragment thereof according to any aspect delineated herein.

In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds arenavirus glycoprotein 1 (GP1), the antibody having one or more complementary determining regions (CDR) selected from

CDR H1 sequence GFTFGTSI (e.g., CR1-07 CDR H1) CDR H2 sequence ISHDESRK (e.g., CR1-07 CDR H2) CDR H3 sequence AKDLSPPYSYAWDIFQYW (e.g., CR1-07 CDR H3) CDR L1 sequence QSVLYSSRSDNKY (e.g., CR1-07 CDR L1) CDR L2 sequence WAS (e.g., CR1-07 CDR L2) CDR L3 sequence QQYYSSPPTF (e.g., CR1-07 CDR L3) CDR H1 sequence GFTFSSA (e.g., CR1-28 CDR H1) CDR H2 sequence IWSDGSNE (e.g., CR1-28 CDR H2) CDR H3 sequence ATDKTYVSGYTSTWYYFNY (e.g., CR1-28 CDR H3) CDR L1 sequence QSIDNW (e.g., CR1-28 CDR L1) CDR L2 sequence KAS (e.g., CR1-28 CDR L2), and CDR L3 sequence QHRT (e.g., CR1-28 CDR L3).

In another aspect, the invention provides an isolated polynucleotide encoding one or more sequences of an isolated antibody or antigen-binding fragment thereof according to any aspect delineated herein.

In another aspect, the invention provides an isolated vector containing the polynucleotide according to any aspect delineated herein.

In another aspect, the invention provides an isolated cell containing the vector according to any aspect delineated herein (e.g., expressing an antibody or antigen-binding fragment thereof according to any aspect delineated herein).

In another aspect, the invention provides an immunogenic composition containing a polypeptide having a TfR1 binding site of a New World arenavirus GP1, wherein the TfR1 binding site includes amino acids 87-235 of JUNV GP1 or corresponding amino acids of an arenavirus GP1.

In another aspect, the invention provides a vaccine containing a polypeptide having a TfR1 binding site of a New World arenavirus GP1, wherein the TfR1 binding site includes amino acids 87-235 of JUNV GP1 or corresponding amino acids of an arenavirus GP1.

In another aspect, the invention provides a method of generating an immune response in a subject involving administering to the subject the immunogenic composition or vaccine according to any aspect delineated herein.

In another aspect, the invention provides an in silico method for identifying an agent that inhibits binding of a New World arenavirus GP1 to a transferrin receptor involving a) generating a three-dimensional representation of a transferrin receptor structural binding pocket using the atomic coordinates of a New World arenavirus surface envelope glycoprotein amino acid residues in the sequence; and b) employing the three-dimensional structure to design or select an agent that inhibits binding, including for example, the atomic coordinates provided at Protein Database (PDB) ID 3KAS; and at PDB ID 5EN2. The atomic coordinates for the antibody CR1-10/JUNV GP1/antibody CR1-28 complex are provided at Protein Database (PDB) ID 5W1K; and the atomic coordinates for the antibody MACV GP1/antibody CR1-07 complex are provided at Protein Database (PDB) ID 5WIM. The atomic coordinates of the protein structure of an unliganded Fab fragment of CR1-07 is provided at Protein Database (PDB) ID 5WIG. The atomic coordinates provided at PDB ID 5W1K and at PDB ID 5WIM were previously described and set forth in ‘pdb’ and ‘txt’ files as Appendices A and B presented in compact discs (CDs), incorporated by reference into the disclosure of priority application number U.S. 62/392,729, filed Jun. 8, 2016, the entire contents of which are incorporated by reference herein.

In another aspect, the invention provides a kit containing the antibody or antigen-binding fragment thereof according to any aspect delineated herein, the polynucleotide according to any aspect delineated herein, the vector according to any aspect delineated herein, the cell according to any aspect delineated herein, the immunogenic composition according to any aspect delineated herein or the vaccine according to any aspect delineated herein.

In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment thereof has one or more complementary determining regions (CDR) selected from

CDR H1 sequence GFTFGTSI (e.g., CR1-07 CDR H1) CDR H2 sequence ISHDESRK (e.g., CR1-07 CDR H2) CDR H3 sequence AKDLSPPYSYAWDIFQYW (e.g., CR1-07 CDR H3) CDR L1 sequence QSVLYSSRSDNKY (e.g., CR1-07 CDR L1) CDR L2 sequence WAS (e.g., CR1-07 CDR L2) CDR L3 sequence QQYYSSPPTF (e.g., CR1-07 CDR L3) CDR H1 sequence GFTFSSA (e.g., CR1-28 CDR H1) CDR H2 sequence IWSDGSNE (e.g., CR1-28 CDR H2) CDR H3 sequence ATDKTYVSGYTSTWYYFNY (e.g., CR1-28 CDR H3) CDR L1 sequence QSIDNW (e.g., CR1-28 CDR L1) CDR L2 sequence KAS (e.g., CR1-28 CDR L2), and CDR L3 sequence QHRT (e.g., CR1-28 CDR L3).

In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment thereof has a heavy chain sequence

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSSAMHWVRQAPGKGLE WVAVIWSDGSNENYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTA VYYCATDKTYVSGYTSTWYYFNYWGQGTLVTVS and a light chain sequence

DIQMTQSPSTLSASVGDRVTITCRASQSIDNWLAWYQQKPGKAPKLL IYTASRLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHRTFGQG TKVEIK or the antibody has a heavy chain sequence

QVQLVESGGGVVHPGRSLRLSCAASGFTFGTSIMHWVRQAPGKGMQ WVAQISHDESRKFYSDSVKGRFTVSRDNSKNTLFLEMSSLRIEDTAVY YCAKDLSPPYSYAWDIFQYWGQGSLVTVS and a light chain sequence

DIVMTQSPESLAVSLGERATINCKSSQSVLYSSRSDNKDYLAWYQQK PGQSPKLLIYWASTRESGVPERFTGSGSGTDFTLSISSLQAEDVAVYY CQQYYSSPPTFGGGTKVELK.

In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment thereof inhibits binding of GP1 and a transferrin receptor 1 (TfR1). In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment thereof binds a TfR1 receptor binding site of GP1. In various embodiments of any aspect delineated herein, the TfR1 receptor binding site includes amino acids 87-235 of JUNV GP1 or the corresponding amino acids of an arenavirus GP1 (see e.g., FIG. 8A). In various embodiments of any aspect delineated herein, the TfR1 receptor binding site includes one or more of amino acids Serine 111, Aspartate 113, Isoleucine 115, and Lysine 216, amino acids 113-124 (JUNV GP1 loop 3), and amino acids 166-174 (JUNV GP1 loop 3) of JUNV GP1 or corresponding amino acids of an arenavirus GP1. In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment thereof the TfR1 receptor binding site interacts with Tyr211 of TfR1. In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment thereof, has a Tyr in CDR H3 that forms a corresponding interaction with GP1 as Tyr211 of TfR1. In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment thereof has neutralizing activity in the subject. In certain embodiments, the antibody is one or more of CR1-07, CR1-28, and GD01.

In various embodiments of any aspect delineated herein, the arenavirus is a mammarenavirus or New World arenavirus. In various embodiments, the New World arenavirus is one or more of Junín (JUNV), Machupo (MACV), Guanarito (GTOV), Sabiá (SBAV), Chapare virus (CHPV), Tacaribe virus (TCRV), or White Water Arroyo virus (WWAV).

In various embodiments of any aspect delineated herein, the isolated antibody or antigen-binding fragment thereof is administered to a subject. In various embodiments, the subject is human. In various embodiments of any aspect delineated herein, the subject is infected or at risk of infection with a New World arenavirus (e.g., Junín (JUNV), Machupo (MACV), Guanarito (GTOV), Sabiá (SBAV), Chapare virus (CHPV), Tacaribe virus (TCRV), or White Water Arroyo virus (WWAV)). In various embodiments of any aspect delineated herein, the subject has or is at risk of having viral hemorrhagic fever.

In various embodiments of any aspect delineated herein, the method is in vivo or in vitro. In various embodiments of any aspect delineated herein, the method is performed in a subject (e.g., a subject in need thereof). In various embodiments of any aspect delineated herein, the arenavirus or arenavirus infection is neutralized in the subject. In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment thereof is administered as plasma or serum. In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment thereof is obtained from blood, plasma, or serum (e.g., from a survivor of an arenavirus infection).

In various embodiments of any aspect delineated herein, the first and second species of New World arenavirus are different species. In various embodiments of any aspect delineated herein, the first arenavirus is naturally occurring or genetically engineered to have enhanced virulence. In various embodiments of any aspect delineated herein, the first species of New World arenavirus is Junín (JUNV), Machupo (MACV), Guanarito (GTOV), Sabiá (SBAV), Chapare virus (CHPV), Tacaribe virus (TCRV), or White Water Arroyo virus (WWAV). In various embodiments of any aspect delineated herein, the second species of New World arenavirus is Junín (JUNV), Machupo (MACV), Guanarito (GTOV), Sabiá (SBAV), Chapare virus (CHPV), Tacaribe virus (TCRV), or White Water Arroyo virus (WWAV).

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By “arenavirus glycoprotein 1 polypeptide (GP1)” is meant a polypeptide or fragment thereof having at least about 85% or greater amino acid identity to one or more of the amino acid sequences provided at GenBank accession nos. EU260463 (CHPV 810419), NC_005077 (GTOV strain INH 95551), D10072 (JUNV MC2), NC_005078 (MACV Carvallo), NC_006317 (SABV SPH114202), NC_004HEK293 (TRVL 11573), or AF228063 (WWAV 9310135) and having arenavirus GP1 biological activity. Exemplary arenavirus GP1 biological activities include binding to transferrin receptor 1, arenavirus cell attachment, arenavirus cell entry, and/or arenavirus infectivity. In various embodiments, the GP1 polypeptide comprises a GP1-receptor binding site comprising residues 87-235 (JUNV numbering; see also alignments in FIG. 8A). An exemplary arenavirus GP1 polypeptide sequence for Junin virus (JUNV) is provided below:

1 dlpllctlnk shlyikggna sfkisfddia vllpeydvii qhpadmswcs ksddqiwlsq 61 wfmnavghdw yldppflcrn rtktegfifq vntsktgine nyakkfktgm hhlyreypds 121 cldgklclmk aqptswplqc p

By “arenavirus GP1 polynucleotide” is meant a polynucleotide encoding an arenavirus GP1 polypeptide. An exemplary arenavirus polynucleotide sequence for Junin virus is provided at GenBank Accession No. D10072, which is provided below.

1 tgcagtaagg ggatcctagg cgattttggt aacgctataa gttgttactg ctttctattt 61 ggacaacatc aaaccatcta ttgtacaatg gggcaattca tcagcttcat gcaagaaata 121 cctacctttt tgcaggaagc tctgaatatt gctcttgttg cagtcagtct cattgccatc 181 attaagggtg tagtaaacct gtacaaaagt ggtttgttcc aattctttgt attcctagca 241 ctcgcaggaa gatcctgcac agaagaagct tttaaaatcg gactgcacac agagttccag 301 actgtgtcct tctcaatggt gggtctcttt tccaacaatc cacatgacct gcctctgttg 361 tgtaccttaa acaagagcca tctttacatt aaggggggca atgcttcatt caagatcagc 421 tttgatgaca tcgcagtgtt gttaccagaa tatgacgtta taattcagca tccggcagat 481 atgagctggt gttctaaaag tgatgatcaa atttggctgt ctcagtggtt catgaatgct 541 gtggggcatg attggtatct agacccacca tttctgtgta ggaaccgtac aaagacagaa 601 ggcttcatct ttcaagtcaa tacctccaag actggtatca atgaaaacta tgccaagaag 661 tttaagactg gtatgcacca tttatataga gaataccccg actcttgctt ggatggcaaa 721 ctgtgtttga tgaaggcaca acccaccagt tggcctctcc aatgtccact tgaccatgtc 781 aacacattac atttcctcac aagaggcaag aacattcagc ttccaaggag gtctttaaaa 841 gcattctttt cctggtctct gacagactca tccggcaagg acacccctgg aggctattgt 901 ctagaagagt ggatgctcgt tgcagccaaa atgaagtgtt ttggcaatac tgctgtagca 961 aaatgcaatc tgaatcatga ctctgaattc tgtgacatgc tgaggctttt tgattacaac 1021 aaaaatgcta tcaaaacctt aaatgatgaa actaagaaac aagtaaatct gatgggacag 1081 acaatcaatg cgctgatatc tgacaattta ttgatgaaaa acaaaattag ggaattgatg 1141 agtgtccctt actgcaatta cacaaaattt tggtatgtca accacacact ttcaggacaa 1201 cactcattac caaggtgctg gttaataaaa aacaacagct atttgaacat ttctgacttc 1261 cgtaatgact ggatactaga aagtgacttc ttaatttctg aaatgctaag caaagagtat 1321 tcggacaggc agggcaaaac tcccttgact ttagttgaca tctgtttttg gagcacagta 1381 ttcttcacag cgtccctctt ccttcacttg gtgggcatac ccacccatag gcacatcaga 1441 ggcgaggcat gccctctgcc ccacaggcta aatagcttgg gtggttgcag atgtggtaag 1501 taccccaatc taaagaaacc aacagtttgg cgcagaggac actaagacct cccgaaggtc 1561 cccaccagcc cgggcattgc ccgggctggt gtggcccccc agtccgcggc ctggccgcgg 1621 actggggagg cactgcttac agtgcatagg ctgccttcgg gaggaacagc aagctcggtg 1681 gtaatagagg tgtaagttct tcttcataga gcttcccatc caacactgac tgaaacatta 1741 tgcagtctag cagagcacag tgtggctcac tggaggccaa cttaaaggga gcatccttat 1801 ctctcttttt cttgctgaca accactccat tgtgatgttt gcataggtgg ccaaatttct 1861 cccagacctg ttggtcgaac tgcctggctt gttctgatgt aagcctaaca tcaaccagct 1921 taagatctct tcttccatgg aggtcaaaca acttcctgat gtcatcggac ccttgagtgg 1981 tcacaaccat gtccggaggc agcaagccaa tcacgtaact aagaactcct ggcattgcat 2041 cttctatgtc tttcattaag atgccgtgag agtgtctgct accattttta aaccctttct 2101 catcatgtgg ttttctgaag cagtgaatat acttgctacc tgcaggctgg aacaacgcca 2161 tctcaacagg gtcagtagct ggtccttcaa tgtcgagcca aagggtattg gtggggtcga 2221 gtttccccac tgcctctctg atgacagctt cttgtatctc tgtcaagtta gccaatctca 2281 aattctgacc gttcttttcc ggttgtctag gtccagcaac tggtttcctt gtcagatcaa 2341 tacttgtgtt gtcccatgac ctgcctatga tttgtgatct ggaaccaata taaggccaac 2401 catcgccaga aaggcaaagt ttgtacagaa ggttttcata agggtttcta ttgcctggtt 2461 tctcatcaat aaacatgcct tctcttcgtt taacctgaat ggttgatttt atgagggaag 2521 aaaagttatc tggggtgact ctgattgtct ccaacatatt tccatcatca agaatggatg 2581 caccagcctt tactgcagct gaaagactaa agttgtagcc agaaatgttg atggagcttt 2641 catccttagt cacaatctgg aggcagtcat gttcctgagt caatctgtca aggtcactca 2701 agtttggata cttcacagtg tatagaagcc caagagaggt taaagcctgt atgacactgt 2761 tcattgtctc acctccttga acagtcatgc atgcaattgt caatgcagga acagaaccaa 2821 actgattgtt aagttttgaa ggatctttaa catcccatac cctcaccaca ccatttcccc 2881 cagttccttg ctgttgaaat cccagtgttc tcaatatctc tgatctcttg gccagttgtg 2941 actgagacaa gttacccatg taaacccctt gagagcctgt ctctgctctt ctaaacttgg 3001 tttttaaatt cccaaggcca gacgccaact ccatccgctc aaccctcccc agatctcccg 3061 ccttgaaaac cgtgtttcgt tgaacactcc tcatggacat gagtctgtca acctctttat 3121 tcaggtccct caacttattg aggtcttctt cccccctttt agtctttctg agtgcccgct 3181 gcacctgtgc cacttggttg aagtcaatgc tgtcagcaat tagcttggca tccttcagaa 3241 catccgactt gacagtctga gtaaattgac tcaaacctct ccttaaggac tgagtccatc 3301 taaagcttgg aacctctttg gagtgtgcca tgccagaaga tctggtggtt ttgatctgag 3361 aaaaaattgc tcagtgaaag tgttagacac tatgcctagg atccactgtg cg

By “transferrin receptor 1 polypeptide (TfR1)” is meant a polypeptide or fragment thereof having at least about 85% or greater amino acid identity to the amino acid sequence provided at GenBank Accession No. AF187320 and having arenavirus GP1 binding activity. An exemplary human TfR1 polypeptide sequence is provided below:

MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENAD NNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTEC ERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLL NENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSA QNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFED LYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAE LSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAE KLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGV IKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDG FQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLG TSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDN AAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVAR AAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGL SLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFL SPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQL ALATWTIQGAANALSGDVWDIDNEF

By “TfR1 polynucleotide” is meant a polynucleotide encoding a TfR1 polypeptide. An exemplary human TfR1 polynucleotide sequence is provided at GenBank Accession No. AF187320, which is provided below.

1 ccggttaggg gccgccatcc cctcagagcg tcgggatatc gggtggcggc tcgggacgga 61 ggacgcgcta gtgtgagtgc gggcttctag aactacaccg accctcgtgt cctcccttca 121 tcctgcgggg ctggctggag cggccgctcc ggtgctgtcc agcagccata gggagccgca 181 cggggagcgg gaaagcggtc gcggccccag gcggggcggc cgggatggag cggggccgcg 241 agcctgtggg gaaggggctg tggcggcgcc tcgagcggct gcaggtacac ggggtcggcg 301 gctgtgcgca gaggcgtccc tgcgcctctc gtcccttcgc ctctcgtccc ttcccttctc 361 tctgccttct tgcccgcctc ctcggtcaca gagcgacgaa tgacgagacc aggtgtaccc 421 cactgtcgct ctcagccccg gggacttcgg gtcctcgccc ttgaaggccg caggccctag 481 tgcgccggct ccgggctgcg ggtccgggag cgcgggcgca gccaaggtgc agctgcgcgg 541 cgtgcggcgc cgggggaaca cgtggctgct ccaggaagtc gccccaggga acggctggga 601 ttcgtgggtg accttgggct cctaaacctt cggttccccg ggctccgggc gggccccgtt 661 ctcactgcgg aaagggacaa agctgtcccc gattcggtta ctagcgtgtt acaggcatta 721 attagatacg ggtttctgta aacttaccct tcggctctgg tacaacctga tcttgccatg 781 ctctgcctgt tcgccttgtg tatggattct gttttctcaa ggcagaatcc gactgaacgc 841 cgcaggtgtt cacattaaga atattgctag cagttctcac tacaagacgg gagccatagg 901 agtgacttat ttgtgaaaga tacttggaga ttagcggtgt ggtcagaggg ctgtgctgga 961 ttgatgggcg ccggagaggc cgattgtgtc acattctgct ggacagttct tttaaggttg 1021 ggagggtggg taagaaaata cattctgatt cggctctttt cggataacgc tttccctctg 1081 cttactgctt gtaagacctc acttacccgg gggactggat tctgcagctt tttccatttt 1141 cttccccgtt gataagagga ttaaagttag gaaattgtat ttggctaccc catgcttata 1201 tgctaagctt actgtgaaaa ttaaggttag gctgtctaga gtacttgtgg accctgctgg 1261 ttccccccgc cccccggccc tgaattttgg gttacaatat gcttctcaag aaaactcaga 1321 gttaagataa tttttgtcat cgattttgca aggttatata tagcatagca tccttcaatt 1381 tcctttggat gttcttatca agtaatggtg gctgtaaacc tagcctgtgt tgaagattgt 1441 tagatagaca gcagtttggc taactccgcc ctaaggcgag gcagttactt gcctctgata 1501 atgtatttaa atgtcatgga gcaagattcc cagctaaacc tgaatcgatc acaatgctag 1561 ttaaaaatga ccctcggctg ggcgcattgg ctcccgcctg taatcccagc actttgggag 1621 gccaaggcag gcggatcacc taaggtcagg agttcgagtc cagtctgact aacatggtga 1681 aatcctgtct ctactaaaaa cacaaaaatt agccgggcat ggtggctcac gtctgtaatt 1741 ccagcacttt gggaggccga ggcaggtgga tcacttgtgg tgaggagttc gagactagcc 1801 tggccaacat ggtgaaaccc cgtctctacg aaaaatacaa aaattagctg ggcatggtgg 1861 tgcacacctg taatcccagc cactcaggag gctgaggctc gaaagtcgct ttaacctggg 1921 aggcagaggt tgtagtgagc tgagatcgtg ctactgcact ccagcctgcg cgacagcgag 1981 atgccatctc aaaaacaaca acaaaaaaac tccctactaa accagaagat gatggggacg 2041 ggaaaagatc ctggtccaat gttttcatta tatttttcat atcatttgga atctcatgca 2101 tcaggcatgc cccagtactg ttaaagacaa tatttttact cattatcagg tagttcaata 2161 ccagttatta caggataggg aagtcagtca gagaaggctt catagagatg aagccttgag 2221 ctgagcctgg aagaaatgag gaggacctca aggaacagag aagaatgttg gtgggtaaaa 2281 acaggggctg gattctgtct gattttggag aaaatagagg gtagtgaagt tgttaggcag 2341 tgaatgtgtt ccatttcatg gttcaaaacg tggggctacg ttttgcctac ctgagcttca 2401 ttattaatgt gagaaattga attgttgttt tcagtcacca ttgagaacac caaagataac 2461 acaagtagct tagattatta tttatttatt tatttttgac agatttcaca cttgttgccc 2521 aggctgtagt gcaatggtgt gatctcggct ccctgcaacc tccacctccc ccaggttcaa 2581 gtgattctcc tgcctcagcc tccccagtag ctgggattac aggcatgcgc caccatgccc 2641 ggctaatttt gtattttttt ggtagagatg ggatttctcc atgtttggtc aggctagtct 2701 caaactctca acctcaggtg atctgctcgc ctcggcctcc caaagtgttg ctgggattac 2761 aggcgtgagc caccatgccc ggccatcgct ttgtgttctt aaaattaatt taaaacaaga 2821 aaacttgagg aatgatggtt cagatgagtg ttaaaacttg caagatgttt tttcctagaa 2881 aaggatgatt aaaattggtt cagggtgggg ccttccagtt ctggctctaa tgattgggtc 2941 cttactgttc tggtggagtg gtagtgataa gctttttgta acagaagggg caaaagattg 3001 tgcttctggc tgggcgcagt ggctcacgcc ttcgatttcc ttgggatgtt cttatcaagt 3061 aactaatctt agcactttgg gaggccaggt gggtggatca ccctaggtca gaagttccag 3121 accagcctgg ccaacgtggt aaaatcccgt ctctactaaa aatacaaaaa ttagctgggc 3181 atggtggtgg gcacctgtaa tcccagctac tcggagtctg aggtgggaaa atggcttgaa 3241 cccgggaggg ggaggttgca gtgagccgag atcctgccat tgcactccag cctaggcaac 3301 aagaccgaaa ctctgtctca agaaaaaaaa aaagattatg cttctaagaa tatggtctat 3361 tgcatatgga cactggtttt taaagcttga atttaaaaag aattttttgt aataggtttt 3421 agagcttttt ctttttcttt tttattattt ttttgagatg gagtctcact ctgtcaccca 3481 ggctggagtg cagtggtgcc atctcggctc actgcaacct ccacctcccg ggttcaagcg 3541 attctcctgc ctcagcctcc ccagtagctg ggattacagg tgcccaccac cacgcccatc 3601 taatttttgt atttttagta gagatggggt ttcaccacct tggccaggct ggtctcaaac 3661 tcctgacctc gtgatccacc tgcctcggct cccaaagtca gtgttgggat tacaggcatg 3721 agccaccgtg cctggccaga aagctttttc ttaatgccag ttataatacc ctttgcttta 3781 caatttgcgt atacctcaca gcttgctttg tgccgtgtgt gtttaatatg ccctaagtgt 3841 actcgtatat aggaaaagct ttaaaatggt atccaataaa tgtccatttt gcaaatctca 3901 ggataatgtc attttcaagt atctagatgc cctcaaacca aattaactgg aaaattcaga 3961 tttcagttaa ggcctttaac ttaacttagg actttgatta cagagatgta ttttgctttg 4021 acaaaagtta aggttaatgt cttaaatttc cagagatgat aacacagttt ctataaccca 4081 gtaacccatt taacttcgct tagattatca attttcagcc ttgaacaggc cacttagttt 4141 ctctaataac tggcctcttc atctgtaaaa cagcgtaatc tgtgtttcat gtttcttctt 4201 gtgtcaaaga tcgaatcact tgtaggaact attcattcag tcaggatcag tatttcttcc 4261 ccttcccatt ggagatggtt tgtggtattt gtagcataac tggtcttgtc tgccttagtc 4321 ttaattttgt ctttttgtat gtgtcacttt cttttttttt tgagacagag ttttgctctt 4381 gttttccagg ctggagtgca gtggcgcgat cttggctcac tgcaagctcc acctcctggg 4441 ttcacgccat tctcctgcct caggctcccg agtggctggg attacaggcg cccaccacca 4501 cgtccggcta atttttgtat ttttagtaga gacagggttt ctccatgttg gccaggctgg 4561 tcttgaactc ctgacctcag gtgatccacc catctcagcc tcccaaagtg ctaggattac 4621 aggtgtgagc caccccgccc agcagtatgt gtcattttgt ctactcagaa catagtggtt 4681 cgtacttata attgcagcac tttgggaggc caaggtggga ggattgtttg aggccaggag 4741 ttcaagacca gcctgggcaa catagggtga ccttgtctct acaaaaagaa aaaaaaagaa 4801 ttatgtatat aacaaacttt aaaaaggatc ccacttaatt tagttgtcat gtaaactatg 4861 gttaaatacc ttttctagaa aagtgataat gtattttaaa aatttaatgt atcatttagc 4921 ctggtaatag aaagctcact aatctgatac agtagtatct ttttcaataa ttctctattc 4981 tgatacctga ggttcttctg tgtggcagtt cagaatgatg gatcaagcta gatcagcatt 5041 ctctaacttg gtaagatatt tcagttgtat ttctgtgtct gcaagaatgt gaaatataca 5101 agcatgactt taactgagta agcatgaaat aataacccca atctattatc aggtattcat 5161 ttacatttga ttggtagtct tgaggcataa ctgatctcac aaagagttca gccttcagta 5221 gggttaatta tggaaagtct tggaagaatt ctcagagtgg ttctgggtgt ggtagacggg 5281 gacagcttag tagataagac gaattaaagc cctgatcaaa atgttttagt aggagaagta 5341 aaaagcaaag gcttgcgggg cgctgtggct cacgcctgta atcccagcac tttgggaggc 5401 tgaggtgggt ggatcacctg tggtcaggag ttcaagacca gcctgaccaa catggtgaaa 5461 ctcggtctct actaaaaata caaaaaaaaa aaaattagct gagtgtcatg gcgcatgcct 5521 gtagtcccag ctacttggga ggctgaggca ggagaattgc ttgaacccgg gaggcggagg 5581 ttgcaatgag ctgagatcac gccattgcac tccagcctgg gcaacagagt gagactccgt 5641 ctcaaaaaaa aaaaaagcaa aggctgaatg aggccaagtg tggtggctca ctcccgcaat 5701 cccagcagtt tgggaggtga ggcaggagga tcacttgagg agtttgagac ttgcctgggc 5761 aacatagcaa gcccatctct attaaaaaaa aatgaaattt taagaaacat tgagtagaca 5821 gtatgagtga aagctgaaag aacatacaaa aaaaacagcc atccaggcca tgacgagaag 5881 gagcctaact cttaacactc tgtctagacc tcaattctct cttatttgta tttatttatt 5941 tatttatttt tattttattt tttgagatgg agtctcagcc tgggctctgt tgcccaggct 6001 ggaatgcagt gtgcaatctt agctcactgt aacctccgcc tcccgagttc aagcgatttt 6061 cctgcctcag cctcctgggt ggctgagatt acaggcgtgc gacaccaagc ccagctagtg 6121 gttttttttt ttgaaatttt agtgagaggg gatttcacca tgttggtcag gcttgtctca 6181 aacttctgac cataaatgat ctgtgtgcct cggccccccc ccagagtgct gggattacag 6241 gcatgagcca ctgtgtctgg tctattctct cttttctgtc ttctatctta tgcttagtca 6301 gaatttctgc ttatttataa ctttgtttta ttcttgctcc cttgctccca ccaacatcat 6361 ggtctgggtt actgcatagt atcctgaact gatttgtgcc cctagtccca gctactcagg 6421 aggctgaggt gggggaatca gtagagccca ggattttgag gctgcagtga gctgtgatca 6481 caccactgta ctccatcctg ggagagagag ggagaccttg tctcaaaaaa aaaaaaaatt 6541 tacaaaaagg gagagactga ctgagaagac cagtgtgaac tagcccttga ctgggcccag 6601 ggaagaatgt tggtttgaaa ggagaacacc ctaaggaata tatagcaagt tatagtaatg 6661 gcccaataac aagcatatac ctaaacgagc ttttcctaaa cattcccgtg cctgttcttc 6721 atcatgcctt tttccgcaac acagtttggt ggagaaccat tgtcatatac ccggttcagc 6781 ctggctcggc aagtagatgg cgataacagt catgtggaga tgaaacttgc tgtagatgaa 6841 gaagaaaatg ctgacaataa cacaaaggcc aatgtcacaa aaccaaaaag gtgtagtgga 6901 agtatctgct atgggactat tgctgtgatc gtctttttct tgattggtga gaatgaccat 6961 tccaaacttc aatgttttct ataaccaact ctgggaagtc tgttatgtca gctcagcata 7021 tagttatttt gtaccttttt tttttttttt tttttggaga cagaggctta ctctcttgcc 7081 taggctagag tgcagtggca ccatgttggc tcactgcaac ctcagcctcc caggtttaag 7141 cgattctctg ccttagcctc ctgagtagct gggactatag gcgcccacga ccacacctgg 7201 ctaatttttt tttttttttt tgagttggag tctcactctg tggctcaggc tggagtgcag 7261 tggcgtgatt tcggctcact gcaatctccg cctcccaggt tcaagcgatt cttctgcctc 7321 agcctcctga ggagctggga ctacaggcgt gcaccaccac acctggctaa tttttgtatt 7381 tttagtagag acggggtttc accatgttgg tcaggctggt ctctaactcc tgacctcgtg 7441 atctgcccgc ctcagcctcc cagagtgctg ggattacagg catgagccac tttcccgtcc 7501 actttatgcc tttttaagtg ccactaggaa gacaatcatg agtagattct atacctgtca 7561 ttgaatttaa tctggttgga aagagagacc catgaattat ttcaatacga cgtgcaagtg 7621 ttttcttttt atttatgtat ttttttgttt gctcccactc caccaggaag caaatgctac 7681 gctagaaaat atccgcagga cactggcggt gcagcagctc agtctcatgt ttcaaggggg 7741 tcttttggaa aaaactacct atgttttgaa taggtagcta tttgaaataa agctagtata 7801 taatttatta agtggtttaa agtatacaaa actgagatgc attatatact ttaaaactac 7861 ttagaatttg atgaaaacca gtattttcaa ggcttacctg aaaatattaa cagattaaca 7921 gggatagatt acaaatgaaa ttctgagttc agctctggaa ctttgtctct ttaggattta 7981 tgattggcta cttgggctat tgtaaagggg tagaaccaaa aactgagtgt gagagactgg 8041 caggaaccga gtctccagtg agggaggagc caggagagga cttccctgca gcacgtcgct 8101 tatattggga tgacctgaag agaaagttgt cggagaaact ggacagcaca gacttcaccg 8161 gcaccatcaa gtgagtgcca gctgctgtgc aagtatctag acaagtaatt caagaattat 8221 gataggccac ggggaaacaa taatggtgac actgtgggga atggcttgtt agagaagaca 8281 agacttgtca tgtttagcta aagaggggaa ggggcctgta aaaaactgaa actatactgg 8341 taggggaaaa ggtagaactt ctctggggag tctccttttt cttaaaaaat taataggccc 8401 ttgatgtttt ccccatcttt gtagcttatt gcttgctgtc attctctggc tctgattttt 8461 cttcttaacc tcttgtaacg ttgtactaag ctctgctttg ctgacaagga catcagtagc 8521 ttttctctgt tttaggaagc aacacatgcc agaagtagcc tttttcatct ccttgatcat 8581 gggtaactgc ctgccttaag tctctagtgc ttcttaattt ctgtgatact cacccattaa 8641 cccctaggcc agtcagtatc cagagacaat gtctccctct ggtggaggct ggaatacggt 8701 ggcgtgctct catctcaccg cagcttcatc cccctaccat agcctcctga gtagctggga 8761 ctataagcac gcagcaccac acccagctaa tttttttttt tttttttaag agatggagtc 8821 tcaccatgtc tcccaggccg gtctgttctt agatttttat ttatttactt atttattttg 8881 agatggagtc ttacgtgttg cccaggctgg agtgcagtgg cacgatttca gctcagtgca 8941 acctctgtct cccaggttta agcaattctc ctacctcagt ctcctgagta gctgagatca 9001 caggcttgta ccaccatgct ctgctaattt ttgtgggttt tttttttgtt tgtttgtttt 9061 ttttttttgc agaaacggga tttcaccatg ttggccaggc tggtgtctaa ctcctgacct 9121 cagatgatcc tcctgtcttg gctcccaaag tgctgggaat taacaggatg gaggcactgc 9181 acctggccaa aatttctaat tttatattta tttatttatt ttaatttgta atgggctttt 9241 ctggcaaaaa ctagaaagca tgctagacaa attctaaaag agctgtaaca cttgtccata 9301 gagttttact atctttgcac atacatgcct ctgtgcaaag aataaattta tcttttcctt 9361 tttgcaagct tcctacactt acagtggtac tataattctt tagtcttaag ttatggcatt 9421 ttctacttca tctagttgat gcttttgtgt ttatttactg tacaccagac cttatggtta 9481 aagtgagagt atagtccacc ccacaaaaat gtaagaacta atatgctgag taaatagaaa 9541 gaagtatggg agatcggacc aaggagcagt tctgcttcag ggtattcaga tgcttcacaa 9601 gtaggtgaga attgaaacta tgcactctgt tcagcacagt attactatct tagattttat 9661 agtagatctg gaagctactc atttccccta aaactatcct aagcagatac attgtgttat 9721 gatgaggctc ttgaaggagt gctttgtagt gggtaaagaa gctttgaagt tacaagattt 9781 ggggcaaaat tttttttctt ttgtgatatt ctggtcagtc tagaagtttt gggtcacagt 9841 tcatttatct gaactatgag ggataatatt ttgctttcaa aacaatttgt ttaatccttg 9901 acttaaatgt ttattttaaa gtaaacttaa gttcatttaa aaggcatttt ctcctccagg 9961 ctgctgaatg aaaattcata tgtccctcgt gaggctggat ctcaaaaaga tgaaaatctt 10021 gcgttgtatg ttgaaaatca atttcgtgaa tttaaactca gcaaagtctg gcgtgatcaa 10081 cattttgtta agattcaggt caaagacagg tatgttgaaa gatggtaaac ttatttttat 10141 acaagtagct attttcaggt gtgctaaata tagcaaagac tttttgttag tgttgttggc 10201 ttttttttga aatggagtct cgctctgttg cccaggctgg agtgcagtgg cacaatctct 10261 gctcactgca gcctttgcct tctgggttca agtgattctc ctgccttagc ctccagagca 10321 gctgggatta caggcgcacg ccaccacgcc cagctaattt ttgtattttt agtagaggtg 10381 ggatggtctc gaacttttga cctcaggtga tcagcccgcc tgggcctccc aaagtgctgg 10441 gattacaggc gtgagccacc gcatgcggcc caaaggcttt taaaataaaa atcaagctta 10501 agatttagag gtaaatttcc tcaagccaaa taatgcaaga catactaaat gtaagctatt 10561 gtgttttttg gaaggatgac cttaggctta ttttaactta atctttttaa cagcgctcaa 10621 aactcggtga tcatagttga taagaacggt agacttgttt acctggtgga gaatcctggg 10681 ggttatgtgg cgtatagtaa ggctgcaaca gttactgtaa gtaaggcaaa acagtgcatg 10741 agactcttcc ctattgaatc attcaaaact catcttttct gttcttagga gttataaatt 10801 tacctgtaaa atgtaaatga tcatgagata ttttggtttt caaccctcta atgacacagt 10861 caacatgcat tgtcttctct ctctaatcac tttccccatg tcctgtttta ttttttcttt 10921 atagtactgc tagcagctga cattatctat gcctttgttt ccattagaat gtgagccaga 10981 tgaagaatca tgggttagtt ctatttactg ccatgttgca gagctttgga gaatgcctga 11041 catacatagt agtatttgct aaacaaatgc atatcctccc tgtggggaca gatgtaaata 11101 tccatcttgg ctggggccta gtcttagctt gattcgttaa tgcttgatct ttttctattt 11161 ttgttttgag acagggtgtc actctgttgc cctagctgga gtgcagtggc gtgagcttgg 11221 ctcactgcat tctccaccac cacccaggct caagtgagcc tcttgagtag ctgagatgtc 11281 cagttaattt ttatttttta ttttattatt attattatta ttattattat tattattatt 11341 tggtagagac agggttttgc catgttggcc aggctggtct tcaattgaaa ctcctgggct 11401 caggtgattc accaccctca gcctcccaaa gtgctgggat tacaggagga agctgcttcg 11461 cccagccctt gatcttttta aatttaggca aatatagtca aattaacaca gaaatagaga 11521 acagatagat agaataattt tcagcttaaa aatcacattt gtggctgggc gtggtggctc 11581 acgcctgtaa tcccagcact ttgggagtcc aaggtgggtg gatcacctaa ggtcagagct 11641 accagcctgg ccaacatggt gaactctatt aaaaatacaa aaattagcca ggcgtggtgg 11701 tgggtgcctg taatcccagc tacttgggag gctggggcag gagaatcgct tgaagccagg 11761 aggtggaggt tgcagtgagc ggatattgcg ccattgcact ccagcctgag caacaaaagc 11821 gaaattccat ctcaaaaaaa aaaaaatcac atttgtaagt caggcgtatc tctagtggat 11881 acctttcggg gctggtgcag ttctgtgttg attccttgcc tttctggata cttgtgcttt 11941 atccctttgc ctggctgcct ataagccagg agtttagaga tgggtaggtt gttctgttaa 12001 agaacattga agatttttgt tcaacttcaa gctttctgta ttgagggctt ggttgttagt 12061 gtcagtttat ggctgttcat ctggactctt atgagcttag gaacatcatg acacatctaa 12121 agttggtgta tgcaagtcac tttgttgtaa tagatggtgt tttaattttg ggacagattt 12181 attatcagac ctttcagtta aaacctattt ctatgatgtc ctcaaggcta agattgttcc 12241 aggcctccag tcccatgacc agcctacata ggcctcttta atctaaatgc ccttcagatg 12301 aaaggcagca tgggataaaa gcatgaaaga cataggaggc caaaaatctt gattttattc 12361 caatagctct gacctaaaag ttgtaaagtt ttttagaact catgtctttt ttaggccttg 12421 gcctcctttt atctcttact tgggttgcct gtcttaggtt tgatctataa cactcttctg 12481 tctcaagatt ttagagctgt tccgatacca taatgctcat taaatctaat gtctttgttc 12541 tagggtaaac tggtccatgc taattttggt actaaaaaag attttgagga tttatacact 12601 cctgtgaatg gatctatagt gattgtcaga gcagggaaaa tcacctttgc agaaaaggtg 12661 agtatgagtt attataatat taaatacagt acttttggta tcttactctt caggtaagtg 12721 atatttcttg ttaatattct tacattctga aaatcagcaa tcttaaactc tcataccaga 12781 taatataatt tattaaaatg ctcaacaaat gaagtaagtt ccacttacct aatcagaagt 12841 gcttaagttg aaagctattt ctctgtataa ctcagtttta cgcatataca gaattcagcc 12901 taaaaagcat gtttataaaa agggaaaatt aatagccaaa acatacacaa ggaactagag 12961 tggaacatat caaaagacag tggtgatttc tgcattttta aaaaaagtgg cagggctcac 13021 acctgtaatc ccaacacttt aggaggctga ggtgggagga ctgcttgagc cctggaggtt 13081 gtgagatgtg actcgtgcca ctccagcctg agtgacagtg agactcaaaa aagtggttgt 13141 cttagatggg tttttgttat tttccttttc tttttttttt tttttttgag acggagtctc 13201 ttgtcaccct ggctggagtg cagtggcgtg atctcagctc actgcaacct ccgcctccca 13261 ggttcaagtg atgctcctgc ctcagccttc tgagtagctg ggattacagg tgcccaccac 13321 caccatgcct ggctaatttt tgcattttta gtagagacag ggtttcacga cgttggcctg 13381 gctggtcttg aactcctgac gtcaggtgat ccacctgcct tggcctccca gattgctggg 13441 attacaggtg tgagccacca ctcctggcct ttgttattct tttttttttt tttttttttt 13501 gagagggagt tttgctcttt tgtccaggct ggagtgcagt ggcgcaattt tggctcactg 13561 caacctctgc cttatggttt caagcgattc tcctgcctca gcttcccaaa tagctgggaa 13621 tacaggcgcc agccactgcg cccagttaat ttttgtattt ttagtagaga cagggtttca 13681 ccatgttggc caggctggtc tcgaactcct gagcttgtga tccccctgcc tcagccttgc 13741 aaggtgttgg gattacaggc gtgagccacc actcctggcc cgttactctt attttgcaaa 13801 caagtgagtt aatttttcag gaagcaatta ataaattacc agataaacaa tttgaaatat 13861 ttggaagtat tataagttta gtcctctgta accatttggc ctttagcata atgtccagga 13921 tgtattctca tttcgtatga tcagatcttc attttctgag ctttatatgt tttcttttag 13981 gttgcaaatg ctgaaagctt aaatgcaatt ggtgtgttga tatacatgga ccagactaaa 14041 tttcccattg ttaacgcaga actttcattc tttggacatg tgagttattt cttgagtaaa 14101 tcaccgtttt gagttccttg agttgttctt ggattcctgt attagcagaa atagcactgt 14161 gtcttcctta actgctcttt tttctgggag ggcgttagca gtaagcaaga agatagatta 14221 cattgacttt gaggctttat tatttgttgc taaaagtact ttgtcaatag tgccttgaat 14281 ttgagaattt ccatatgcca tgaaaacagg agtcacattg tagagccact cagatttttg 14341 aggcttcaag ttggtaaact aaggttgggc tgcagcctta taccaaacct gaatcttaca 14401 gagaagtttt gaggaagtat gtgatggagt gaattgctct tttgttttcc taggctcatc 14461 tggggacagg tgacccttac acacctggat tcccttcctt caatcacact cagtttccac 14521 catctcggtc atcaggattg cctaatatac ctgtccagac aatctccaga gctgctgcag 14581 aaaagctgtt tgggtaagtt tttatttgaa agcggtcttg catagtgagg ttttatgatt 14641 agggaagaac ggtaatatgt tgattaaaat gttaaacatg atgataattt tccacatttg 14701 ggaatttgga ggatggttag ctgttacctt ggtacagata taaatgaatt tttctctata 14761 aagagatctt aataatggac tttggactta taaacacagg agaataagga gacttataaa 14821 cccagtgaat tccttgtgca agattcctgg catagcttct gctcaagtac ttctgtgctg 14881 aagcacgatg cctatagcag cccattcagt tagtagagag aatctgttag aattcatatt 14941 gggataaaat cctattcccc gaaacatcca tttgtctgga tttttcttct aagcaatacg 15001 gtatgattcc agttaccttg tctatagatt gagaagcctt gcttcttatg atggtttcca 15061 gagaaaccat ttatggttgt ggctcgctcc tctggatatc ctccctgtaa cgttagtgtc 15121 tctctttaaa aacagagccc agttccatta gagtacgtag cattaatttt gatttggatg 15181 tggacatatt tatcatttcc tgttattaca ggatggagat ctagtttcag gcctttatga 15241 aagcatttat ctcctgtatg acataggttc tctgatcctg tttctttaac ataattggat 15301 agtgaaaata tattctactt gatagtctca aatgaagaac atctgtatat aaggagtata 15361 tgaaatacca tgactgttga tcatgctgag aggcctttag cataggggag tgtgtaacat 15421 accatgacta ttgataacgc taagaggcat ttagcatagg tgattttttt gactcattct 15481 acctaccacc tacaaatcgt aaccaacctg tgaagccagc gtcccatctt atccttatgt 15541 agtaggggag taataatgtt tcaacctata tttatgtaag taagccgcct taggagcagc 15601 tattatttgg gtaacacaga ggaattagat aggggaagca caagattttt tttttatttt 15661 gacagtctca ctctgttgcc caggcaggag tgcagtggca tgatcctggc tcactgcaac 15721 ctccacctcc tgggttcaag gaattctgcc tcggccttcc aaagtgctgg gattacaggc 15781 gtgagccacc gcacccagct gaggaatatt ttttataact gagctaagaa tgtgtactat 15841 ccttgttagt ggtgacagtt gggaaacata aaagtgtatt aatattcttt tatatattag 15901 aagaacttca ttttgagtcc atcttggtat gtattccaaa tataaactac gtaagtatgt 15961 ctggcagaaa ggcatagtta gagaagtgtt ttaaaatatt gcttaattaa tggtttccaa 16021 ttggctgcct gcagatcaaa gtaagacgca aatggttcac cactagagag taagtttttt 16081 tttgttttta ttttgttttg ttttgttttg tttttttttg agacaggact tgtgctctgt 16141 tgcccaggct ggagtgcagt ggcgtgatct cggctcactg cagcctccgc ctcccaggtt 16201 caagcgattc tcctcctcat cctccttagt agctgggatt acaggcgcat gccaccatgc 16261 ccagttaatt tttgtatttt tagtagagtc ggggtttcac catgtcggtc aggctggtct 16321 tgaactcctg accttgtgat ccacctgcct cagcctccca aagtgctggg attacagggg 16381 tgagccactt cgccgggctg agagtaagtt ttgtttatat gtcctcttaa tctgtaactt 16441 cactggacag gagtaaaccc tgggcaagga acaataactc agaacttacg cctgctttct 16501 gattctagga atatggaagg agactgtccc tctgactgga aaacagactc tacatgtagg 16561 atggtaacct cagaaagcaa gaatgtgaag ctcactgtga gcaatgtgct gaaagagata 16621 aaaattctta acatctttgg agttattaaa ggctttgtag aaccaggtaa agaccgcccc 16681 ccccccccgc cccgcttttt tttttgtttt cttttctgtt cctaaggatg tggctagaga 16741 aggagcgagt gtaggaatgc tggcttggct tggttttatg aagtgctcaa tcttgtctgt 16801 cctaaagtta attgtttatg tgttagtttc tttttttttt tttgagaaag agtttcactc 16861 ttgttgccca ggctggagtg cagtggtgtg atctcggctc actgcaattt ccgcctccca 16921 ggttcaagcc attctcctgc ctcagcctcc ccagtagctg ggattacagg catgcgccac 16981 cacgcctggc taattttgta tttttagtag agacagggtt tctccatgtt ggtcaggctc 17041 catgaggtca gagctcctga cctcaggtga tctgcctccc tcagcctccc aaagtgctgg 17101 gattacaggc atgagccacc gcgcccagcc tatgtgtcag ttttattggg aggtaataag 17161 gccctaggaa attcttgtta aaaattgacc attatgacta gaaaatctat tctaagttat 17221 tgactagacc agtgataagc aaatttctta agggggcaga taataaacct tttgccaggc 17281 tatacagtct ctcttttagc tgttcaactc tgtgggagca agaaaacagg cacagactgt 17341 gcgtagtgaa tgggcatgcg ggctgtagtt tgccagtccc tggagttaac tttaaatgca 17401 atcaaacagc caacacttac taagaaataa gtccttggac ttaagtagtc agtaaattta 17461 attagtagtg taggaaaaaa gtagctctac tagtaaggta aaattaatat tctcatgtga 17521 atttttaatt cccagagttt ttagtcagat ttgaagtaag gctcttttat ccttaataca 17581 tatactgtgc ttttaccttt ctactgatgt gctaattcta gaaaagttta gaagctgatt 17641 atataagttc tttcttcctc ttttttcttt tttaaagatc actatgttgt agttggggcc 17701 cagagagatg catggggccc tggagctgca aaatccggtg taggcacagc tctcctattg 17761 aaacttgccc agatgttctc agatatggtc ttaaaaggta gagtacaaat tttgattctt 17821 ttgaatattg gtgcactgca tacagttcta gatgttatac tgtgctttgc tcactttgcc 17881 tgcattcctg tggttctcat gctagactct agttcttaaa tacaaggcag attgtctttt 17941 gttgtagctg ccttttcctt tgaaacagct ttccataaag tgtcttaact gtgctagaaa 18001 tcaccagttt ctttgagaca gtggagttac tgagccctag tgcttagtgt ggtggatcca 18061 gagtgataag gtggatcata gtccctaagc aatctatttg aaagcagtag catacctctc 18121 tactcatgta ctgaggccta acagctattg aaattttgtt ctgtttattc atttattcat 18181 tttttgagac agagtttcac tctgttgccc aggctggagt gcagtggtgt gatctcactt 18241 agctcactgc aacctctgcc tcccgacttc aagcaattct cctgcctcag cctcctgagt 18301 agctgggatt acaggcaccc acaaccacac ccggccaatt tttgtatttt tactagagac 18361 agggtttcgc catgttggcc aggctagtct cctgacctca agtgattcac ccgcctcagc 18421 ctcccaaaat gctgctagtg agccaccgtg ccctgcctct tttgtttatt tttgagcctt 18481 tctgtcctac tttccactct ttcacactcc tacccaccca cacattcaaa atcacgtcac 18541 attcttgaat ctttgggtct tagggcaaac tgcaatgctt aactttaata cagagtacat 18601 taatggagaa aaagatacag tgagggaagt gggagggatg gagggcagtg tgatacatgt 18661 aacctcaaaa ggtgctgtca caaatatagt ttctctgcaa actcttcttg taacttaaat 18721 ttgtgaattt ttattttatt ttagaattat ttttttaaaa atccaatgtt tggatttacc 18781 tctgaagatt tttctcaaga tgtgcatcat catgcttaca ctgttttcaa tgatctctgg 18841 gtccagaagt taaggaactc caagaatgaa attgtgaaag ggtgactact ggcccctgct 18901 atgtactcaa aatcttattt gcagatctcc tatcttgctc tttgtaggtt acttaaaaaa 18961 aaattattcc tattctttta ttcttttacc tggggtctca aaaaaagatt ttgttagttt 19021 ttttttttct tttttggctc agaaaaaaaa gattttggaa tctgctatct tggaattaga 19081 ccttctgatt catcttaagt gggatacctt ccatccttgt ctgtgtataa cctttcctca 19141 ggtaaagcta acttttttct ctttcagatg ggtttcagcc cagcagaagc attatctttg 19201 ccagttggag tgctggagac tttggatcgg ttggtgccac tgaatggcta gaggtattct 19261 ttatcatccc ttcccatatt ggacacgagc ttgtgggctt aggctgttgt ccagaagtga 19321 tgtttttata ggtttgattt taccactttt gcctttgcgt ttagtctcag tagagtccag 19381 aattgaaaat gaatccctaa tgctactgta tgtgataaat aaacagattt atacttatta 19441 gtgttttccc ttctcttcta gggatacctt tcgtccctgc atttaaaggc tttcacttat 19501 attaatctgg ataaagcggt tcttggtaag tatccctttc attagctgtt tatgaattca 19561 ggtaaacttt tttgagatgg agttttgctc ttgtcgccca gactggagtg caatggcacg 19621 atctcggctc actgcaacct ctgtctccca gattcaagcg attctcctgc cttagcctcc 19681 tgagtagctg ggattacagg tgctcactac cacacccagc taattttttg tatttttagt 19741 agagacaggg cttcactatg ttggccagat ggtctcgaac tcctgacctc aggcgatctg 19801 cctgccttgg cctgggatta cagatgtggg ccactgtgcc tggccagata aactacttga 19861 agtggaagaa agcttttttt tttttgagac agagtctcac tgtattgccc aggctagagt 19921 gcagtggcac aatcttggct tcactgcagc cttgacctcc tgggctcagg tgatcctccc 19981 acttcagcca cctggctaat tttttttgta gagatggggt tttgccatgt tgcccaggct 20041 ggtgtcaaac tcctgagttc aagcattcta tgtcagctgc ccaaaatgct gcggttacag 20101 gcatgagcca ttgccctcag cctgtatctt aaccttcctt taaatagtct gtcaagttac 20161 acagtgagca caattgcttg tctagaacag tgggtagttc tcagtgtggc ccccagatga 20221 gtagcattag gaactgttac gaaatgcaaa ctgtcatgtc taccccagac ctttgagtca 20281 gaaatgggag tgttggtcta aaaactgggc tttttgggct gggcgcagtg gtgcacgcct 20341 gtaatcccag cacttgggag gccaaggcag gcggatcacc tgaggtcagg agttcgagac 20401 cagcctggcc aacatggtga aaccccgtct ctactaaaaa taccaaaatt agccgggtgt 20461 ggtggcgagt acctgtaatc ccagctactc aggaggctga ggtaggagaa tcacttgaac 20521 ccgggaggca gaggttgcag tgagccaaga tggcgccatt gccctccagc ctgggcgaca 20581 gagcgagact cctctcaaaa aaacaaacaa aaaaactggg cttttttttt ggcaaccctt 20641 ggggaataaa aacctattat tttcttaagg acaaagtatc ctgaagcaaa aaaacccaaa 20701 caaagaaaca aaaaacttta acaagcacta caggtaattc tgatggacta aagttttagg 20761 accataagtc tggactatat tgaggtgaga agaaactaaa ctatgccata tagaatggta 20821 cttagagagt aattcacatc ctgttacgtt gtggcatcac tgatagaaat attggataat 20881 gaaacttcta gaagagtttg aatgttcact ggcagcagaa aattgacaaa agggtttgaa 20941 tgttatttaa agtgcagctg tccattcaac aggaatgggg taaaaaagaa agtgcagatg 21001 tagctgatga gctgagaata gtgaaatgtc tacatggggg aaaaaaggaa agagttacaa 21061 ttaaacctct ctaggttagt tatttccctg ttgtatgttt gccgcagaat gtgctgagta 21121 tagcaagcat actatgtata gctctaacct gggtgaacca gaaagttaaa catgaaattg 21181 ctaatggggg aggctgcaca tgtgtggggg cagtggattt atgggacctc taggtacctt 21241 tctcttaatt ttgctgctga acctaaaact ggtctgattt tttttttttt tttttttttt 21301 tttgagatgg agtctcgctt tgtcacccag gctggagtgc agtggcgcca tcccagctca 21361 ctgcaagctc cgcctcccgg gttcatgcca ttctcctgcc tcagcctccc gagtagctgg 21421 gactacaggt gcccgccacc acacccggct aattttttgt atttttagta gagacggggt 21481 ttcaactgtg ccagccaaga tggtctcgat ctcctggacc ttgtgatcca cccgtctggg 21541 cctcccaaag tcctgggatt acaggcgtga gccaccgtgc ccggcctaac tgctctgata 21601 tttttaaaaa aggtgacttg gattaaagta tgcaaactca aggagtagta cgagcccact 21661 tgagtgaagt tagcttagtt gctaaagagc ttgataccaa aattattttg tttattgatg 21721 aatgggatag ttgttgcaca gggccactga tctagattac tgtcttattt gtcaagtact 21781 tatagttgta gaagtagcag tgtgaaaatt atcggaatgg agaaggggca atgataaaac 21841 aatttatttt caggtaccag caacttcaag gtttctgcca gcccactgtt gtatacgctt 21901 attgagaaaa caatgcaaaa tgtgagtata tacctcatta caaaaatgta tgacttaatt 21961 tttgttgaat caacctgaga taaaaaacac tgatatgtaa accgtagtca gtaacaaaaa 22021 ataggaattg agaataaatt ttatagcagc gttatttaag ggatacttgc ctattgaacc 22081 atatgagatg agggctccaa tcttaaagaa tatgttgttt atttaggaat atataacaaa 22141 atgccatgag gcctaagctg tagtgcaggg gcccacaggg gattgtctat agagtcacaa 22201 tgctaaggaa ggcttaaatc aacttgaact atactttgag aaggccggag gaaatattca 22261 ttgaataaac aattaccata gttttgaatg agggagagca tgtgaaagaa aatatttgcc 22321 tatgtggggt ggggggaggg gagagggata gcattgggag atatacctaa cgctagatga 22381 cgagttagtg ggtgcagtgc accagcatgg cacatgtata catatgtaac taacctgcac 22441 aatgtgcaca tgtaccctaa aatttaaagt ataattaaaa aataaataaa taaataaata 22501 aaaaagaaaa tatttgccta atgggattag aggctgtata ttggagtgag taaagttaga 22561 aacaggtcag atagcagaca ttggccttga tccaagagat actagggatt ataggatctc 22621 aagcaaggtg atcatgaatt tgccagttag gttggtggca taagatgcac tagaaggaag 22681 aaaccagaaa caagataggc atagtgtgaa gcccagagag ggcttttaca attgtgggta 22741 agccaccatg gttttgtttg tttgtttgtt tgtttttgag atggagtctt cctctgtctt 22801 cctccagcac tcaggctgga gtgcaatggt gcgatcttgg ctcactgcag cctccacctc 22861 ctgggttcaa gcgattctcc tgcctcagcc tcctgaggag ctgggactac aggtgcacac 22921 caccacacct ggctaatttt tgtattttta gtagagacgg ggtttcacca tgttggccag 22981 gatggtctca atcccttgac ctcatgatct cttgaccttg gcctcccaaa atgctgggat 23041 tacaggtgtg agccaccgcg cccagctttt attcattttt ttttcctttt ttttttttaa 23101 gagataaagt cttgctgtgt tgcccaggct agtctcaaac ttctgggctc aagtgatcct 23161 cccagctcgg cctcccaaag tgttgggatt gcagttgtga actaccccac ccggcctagg 23221 cccactttta aaatgttaat taagaagaaa acattttttc ctcaccataa ctaaaggaac 23281 tgacaacatg agcctagtta gtaataactt aaagttgaga ttgttttaac tatcataaat 23341 aatttctttt caagaagata taatacaagt tttttattta aaaattgtta tagagtaaca 23401 tgttagtgac tagcctgaat aaataagttc caaaaggtta gttttaatga taatttctta 23461 tcttacaggt gaagcatccg gttactgggc aatttctata tcaggacagc aactgggcca 23521 gcaaagtgta agttgagaaa agtgaatgaa caaactaata gaaaagcaga gattctacct 23581 actacattag gtagaatcta aatctgtcct tgcattgaac ttacttacac ctaaagatat 23641 tcagctaaag aatttatttt ggatgggggg actagcacga tagaacagtc tattctttaa 23701 aacactttct aaagacacat tctttgtctt tgcagtgaga aactcacttt agacaatgct 23761 gctttccctt tccttgcata ttctggaatc ccagcagttt ctttctgttt ttgcgaggta 23821 agtctgttca tttaaatgac aaatggagag aggctctcta aaaggaagtg atctgtattt 23881 gggaataggg cattgcaatg ggaatgcctg tgctatagtc aactatgtac atattcagag 23941 gagtaaagga aaacaagttt ttaaggacaa atgatgagga ttacaaaatt actttgaggt 24001 aattattctt ggctaccaag atcaataaca aggatgacac cagtccaagg ttgacaggca 24061 gttgctaggc agattttctc acgagagaag ttttttggtg caaggttgca gtggcctttg 24121 tgtaaggttg tagtttttgt aaaaaggaaa aaaaaaccct taatccttgt tatcagacat 24181 acaagggtga gacccttctc ttcagggttg catttttgtt aacactagtg actccatttt 24241 gattttgaca acttgcacat atcttagttg ttgggttttt tttgtttgtt ttttgttttg 24301 tttttttttt ttttttgaca atggtgactt gctctgtcac ccaggctggg gttgcagtgg 24361 cccgatcatg gctcactgca gcctcaagca gtcctccccc ttcagcctcc aaactgtttg 24421 gattataggc aagagctact tactacacct ggccatggat cttcttgtgt caactttgag 24481 accaaattta ctgcagtgct taaaatgttt taagaattat acacatgtgc tggatgtggt 24541 agctcacgcc tgtaatcctg tagcaggaca agccgcagac aaatcccctc agacaccgag 24601 ttaaagaagg aagggcttta tttggctggg agctttggca agactcacgt ctccaaaaac 24661 tgagctcccc gagtgagcaa ttcctgacct ttttaagggc ttacaactaa gggagtctgc 24721 gtgagagggt cgtgatcaat tgggcaagca gggggtacat gactgggggt tgcatgtacc 24781 ggtaattaga acagaacaga acaggacggg attttcacag tgcttttcta tacaatgtct 24841 ggaatctata gataacataa ctggttaggt cagggctcga tctttaacca ggtccagggt 24901 gcggcagcgc tgggctgtcc acctctgcct tttagttttt acttcttctt tctttggagg 24961 cagaaattgg gcataagaca atatgagggg tggtctcctc ccttaatccc agcactttgg 25021 gaggccaagg aaggcggatt acgaagtcag gagtttgaga ctagcctgac caaaatggtg 25081 aaaccccgtc tctgctaaaa atacaaaaat tagtcggaca tagtggcgtt gtgcctgtaa 25141 ccccagctac tcaggaggct gagcaggaga atcgcttgaa cctgggaagc agagttgcag 25201 tgagcctgag aatggaccac tgcactgcag ctggggttta gggtgacaga cgcttgggtg 25261 acagagcaag accctgtctc caaaaaaaaa agttatacac atgtaattat tgcatgttcc 25321 ttcattatta gttttaacaa ctagacttgt taatctcaat agcttaatta gcatttgagt 25381 ttattgctaa aattctttat gagttttaat aatgaggctt ggccgagtgt ggtgggtcat 25441 acctgtaatc ccagcatttg ggaggccaag gcgggtagat cacttgaggt caggagttca 25501 agaccagcct ggccaacatg gttaaacccc atctctacta aaaatacaaa aaaattagct 25561 gggcatggtg gcacatgcct ataatcccag ctgctcagga ggctgaggtg gaagaatcgc 25621 ttgaacccag ggggcagagg ttgcagtgag ccgagatcgt gccattgcac tctagcctgg 25681 gcaacagagc aagatcgtct aacaacaaca acaaaaaaac caaggctgaa tttcttgagt 25741 gattgagcag tggctatcta ttggacagtc cagctgaaca gtatttttcc tcaggctggg 25801 tgcagtaact ctcacctata ttccggcaca ttgggtggct gaggtgggca gatcacttga 25861 ggccaggagt gagaccagcc tgggtaacgt gcctagacta tatctctaca aaaaattttt 25921 taaataaagc tggcccagtg gccagctgta gtcccaactc cttgggaggc tgaggcaggg 25981 gggtcacttg agctaggagc ttaaggctgt ggggagccat gattgcatca ccacgctcca 26041 gcctgggtga cagagtgact cgtctcaagg ctgcagggag ccatgattgc atcactgcac 26101 tccagcctgg gtgacagaga gaggcctcgt ctcaaggctg cggggagctg tgattgcatc 26161 actgcactct agcctgggtg acagagtgac ctcgtctcaa ggctagaggg agccatgatt 26221 gcatcactgc actccagcct gggtgacaga gagagacctc gtctcaaggc tgcagggagt 26281 catgattgca tcgctgcact ctagcctggg tgacagagac cttgtctcaa ggctgcgggg 26341 agtcatgatt gcattgctgc actctagcct gggtgacaga gagaccttgt ctcaaggctg 26401 tggggagtcg tgattgcatc actgcactct agcctgggtg acagagagac ctcgtctcaa 26461 ggtatgtttc atgtatcttc tcttttttca ttgataaagg cccaaacttc ccagagagaa 26521 aaacatacag ccttaaggaa tttggctaga agtctattca gggcatatga tacgatagaa 26581 cagggattct gtagaacctg gaagaaagta gtcaactcta ggagtaggtt agcttgagag 26641 aagtagcaag aatgtactta aagcagcaga taatgagata gaattggggt aaattgcggt 26701 aggaatatgt tagaagcaag ggtggaactg tcgtcactgt tacctcgatg gcgaagccag 26761 aatgtgaggc tcttgctctt agaactcacg tgagtaccat agcctcgcat tgtctcacag 26821 gacacagatt atccttattt gggtaccacc atggacacct ataaggaact gattgagagg 26881 attcctgagt tgaacaaagt ggcacgagca gctgcagagg tcgctggtca gttcgtgatt 26941 aaactaaccc atgatgttga attgaacctg gactatgaga ggtacaacag ccaactgctt 27001 tcatttgtga gggatctgaa ccaatacaga gcagacataa aggtgagcac tgattccaat 27061 tacgttttta ttttgctgaa tgtcaagtat tttgaaatgt gatgtgttcc tgtgtgttcc 27121 tgttggaagg gtgattgtag ccatagtaca ttttaaagtg aactgaggta taattgtatg 27181 tagaattggt aacttgtttg agagaagtcg ggaggctgtg gattagagac ctaggacaga 27241 gctcagcagg tgtttcagaa tccagagcag tgtcaggttt tctgtcactc atgtctccca 27301 ggcagcccgt cagtaggaca cggaatatga agatctcagc aaggagttgg gctgtgtgcc 27361 tctcgggcgt gacccggatg gaaagacagc acagctagca ggattccatc tcgtagtgat 27421 ctgcgcatct aaaagtcaaa tattctatta aacgaaactg ataagcaggg tgaggtggtg 27481 catacccgta gccccagcta cttctgctga ggcaggagga ttgctggagt ccagcccagg 27541 caacatagca aaatcccatc tctaaataaa ttaataaaac taatattaaa gtagcttcca 27601 gattgtttta tggtactagg agttgatttt taacagatct cttaattgaa gtaaatcact 27661 gacaaccgaa tctttttata tcttttttat ttttattttt attttatttt ttttgagaga 27721 tgaagtctcg ctcttgtccc ccaggctgga atgcaatgac atgatctcgg ctcactgcaa 27781 cctccacctc ccggctttaa gcgattctcc tgcctcggct ccccaggtag ctgggattac 27841 aggcgtgtgc caccatgccc agatagtttt tgtgttttta agtagaagcc ggggttttac 27901 catgttggcc aggctgaagt gcagtggcga gatctcggct cactgtaaga tgcgcctccc 27961 gggttcacgc cattctcctg cctaagcctc ccgagtagct gggactacag gtgccggcca 28021 ccacgcccgg ctaatttttt gtatttttag tagagccggg gttttaccat gttggccagg 28081 ctggtcttga actcctgacc tcaggtgatc cacccacctc ggcctcccaa agtgctggga 28141 tcacaggcat gagccaccac gcccggcatc ttatgtcttt cttgaaacta attgtaactg 28201 ttgaaaatgg aacttaccag gtggaaataa ctaaatcctg aagtaccttg aaccaaaatg 28261 ttttccccta tagggacatt ttcctccaaa aggagaattg aactggaaac aatatagtac 28321 ataggattat ataattatgt tcaatttctt aatgagaatg gttttcttac atgctgggct 28381 caaatatgag tgtatatcac agtccataga gcttggaacc cctgtgcaag gtgctttcgg 28441 agtcttgagc ttatctgcga gttcctttta tcagaatctt acttaacgca cgttaaatta 28501 gaaaggcata caaaagaatg tccttagaaa taaaacttct catagcgaat aatgtctgtt 28561 tcaggaaatg ggcctgagtt tacagtggct gtattctgct cgtggagact tcttccgtgc 28621 tacttccaga ctaacaacag atttcgggaa tgctgagaaa acagacagat ttgtcatgaa 28681 gaaactcaat gatcgtgtca tgagagtaag tgaacttttg ggaaaggagg aactaaagta 28741 tgtgtaaaat aaccgataaa tcttacactt ctgcaaagtg gacaaactct aggagtctag 28801 aattccttta agaagggagc attaatggtt tagctgtcat tttctgtttc tgctgtctaa 28861 ttcagaactt agtcaaacct agtctttttg gaagagactt gctgtaaaac ttccatgtat 28921 gctccaatgg ggaaaagatc tgaacacatt taaagttttc ctttgtaaaa tgaatcagtt 28981 tcctttaaaa aaaatttttt ttttttgaga cagagtttca ctgttgttgc ccaagctgga 29041 gtgcaatggc acggtctcgg ctcactgcac cctccacctc ccaggttcaa gtgattctcc 29101 tgccttagcc tcccgagttg ctgtgattac aggtgcccaa caccacgccc ggctcatttt 29161 ttgtattttt agtagaaacg gagtttcacc atgttagcca ggccggtctc gaactcccaa 29221 cctcaagtga tccacctgcc ttggcctccc aaagtactgg tattacaagc gtgagccgct 29281 gtgcccagcc tcctttagaa ttttaacctt agaagattag cattagcctg attctcagca 29341 ttcttttttc cttactctgc tatagaaagt ctgatcagct ggctgggtac agtggctcat 29401 gcctgtaatt ccagtacttt gggaggccga ggcaagcgga tcacctgagg tcaggagttc 29461 aagaccagcc tgaccaacat ggagaaaccc catctctact aaaaatacaa aaattagctg 29521 ggcgtggtgg tgcatgcctg taattccagc tgctcaggag gctgaggcag gagaattact 29581 tgaacccggg aggtggaggt tgcagtgagc tgagatcgcg ccattgtact ccagcctggg 29641 caacaagagc gaaactctgt ctcaacaaca acaaaaagcc gggcacggtg gctcacacct 29701 gtaatcccag catgaattgc ttgaactcgg gaggtggagg gtaccagtga gccgagatag 29761 cgctgttgca ctccagtctg ggcaacaaga gcgaaactct gtgtcaaaaa aaaaaaaaaa 29821 aaaaaagtct gatcggcatt cttaaatttg ggacatttta catttgaagt gaactgttgt 29881 tttactacaa aagtcacagg gctgtgtaaa ttgccttgtg tgttgttttc gtaggtggag 29941 tatcacttcc tctctcccta cgtatctcca aaagagtctc ctttccgaca tgtcttctgg 30001 ggctccggct ctcacacgct gccagcttta ctggagaact tgaaactgcg taaacaaaat 30061 aacggtgctt ttaatgaaac gctgttcaga aaccagttgg ctctagctac ttggactatt 30121 cagggagctg caaatgccct ctctggtgac gtttgggaca ttgacaatga gttttaaatg 30181 tgatacccat agcttccatg agaacagcag ggtagtctgg tttctagact tgtgctgatc 30241 gtgctaaatt ttcagtaggg ctacaaaacc tgatgttaaa attccatccc atcatcttgg 30301 tactactaga tgtctttagg cagcagcttt taatacaggg tagataacct gtacttcaag 30361 ttaaagtgaa taaccactta aaaaatgtcc atgatggaat attcccctat ctctagaatt 30421 ttaagtgctt tgtaatggga actgcctctt tcctgttgtt gttaatgaaa atgtcagaaa 30481 ccagttatgt gaatgatctc tctgaatcct aagggctggt ctctgctgaa ggttgtaagt 30541 ggtcgcttac tttgagtgat cctccaactt catttgatgc taaataggag ataccaggtt 30601 gaaagacctt ctccaaatga gatctaagcc tttccataag gaatgtagct ggtttcctca 30661 ttcctgaaag aaacagttaa ctttcagaag agatgggctt gttttcttgc caatgaggtc 30721 tgaaatggag gtccttctgc tggataaaat gaggttcaac tgttgattgc aggaataagg 30781 ccttaatatg ttaacctcag tgtcatttat gaaaagaggg gaccagaagc caaagactta 30841 gtatattttc ttttcctctg tcccttcccc cataagcctc catttagttc tttgttattt 30901 ttgtttcttc caaagcacat tgaaagagaa ccagtttcag gtgtttagtt gcagactcag 30961 tttgtcagac tttaaagaat aatatgctgc caaattttgg ccaaagtgtt aatcttaggg 31021 gagagctttc tgtccttttg gcactgagat atttattgtt tatttatcag tgacagagtt 31081 cactataaat ggtgtttttt taatagaata taattatcgg aagcagtgcc ttccataatt 31141 atgacagtta tactgtcggt tttttttaaa taaaagcagc atctgctaat aaaacccaac 31201 agatcctgga agttttgcat ttatggtcaa cacttaaggg ttttagaaaa cagccgtcag 31261 ccaaatgtaa ttgaataaag ttgaagctaa gatttagaga tgaattaaat ttaattaggg 31321 gttgctaaga agcgagcact gaccagataa gaatgctggt tttcctaaat gcagtgaatt 31381 gtgaccaagt tataaatcaa tgtcacttaa aggctgtggt agtactcctg caaaatttta 31441 tagctcagtt tatccaaggt gtaactctaa ttcccatttt gcaaaatttc cagtaccttt 31501 gtcacaatcc taacacatta tcgggagcag tgtcttccat aatgtataaa gaacaaggta 31561 gtttttacct accacagtgt ctgtatcgga gacagtgatc tccatatgtt acactaaggg 31621 tgtaagtaat tatcgggaac agtgtttccc ataattttct tcatgcaatg acatcttcaa 31681 agcttgaaga tcgttagtat ctaacatgta tcccaactcc tataattccc tatcttttag 31741 ttttagttgc agaaacattt tgtggcatta agcattgggt gggtaaattc aaccactgta 31801 aaatgaaatt actacaaaat ttgaaattta gcttgggttt ttgttacctt tatggtttct 31861 ccaggtcctc tacttaatga gatagtagca tacatttata atgtttgcta ttgacaagtc 31921 attttaactt tatcacatta tttgcatgtt acctcctata aacttagtgc ggacaagttt 31981 taatccagaa ttgacctttt gacttaaagc agggggactt tgtatagaag gtttgggggc 32041 tgtggggaag gagagtcccc tgaaggtctg acacgtctgc ctacccattc gtggtgatca 32101 attaaatgta ggtatgaata agttcgaagc ttcgtgagtg aaccatcatt ataaacgtga 32161 tgatcagctg tttgtcatag ggcagttgga aacggccttc tagggaaaag ttcatagggt 32221 ctcttcaggt tcttagtgtc acttacctag atttacagcc tcacttgaat gtgtcactac 32281 tcacagtctc tttaatcttc agttttatct ttaatctcct cttttatctt ggactgacat 32341 ttagcgtagc taagtgaaaa ggtcatagct gagattcctg gttcgggtgt tacgcacacg 32401 tacttaaatg aaagcatgtg gcatgttcat cgtataacac aatatgaata cagggcatgc 32461 attttgcagc agtgagtctc ttcagaaaac ccttttctac agttagggtt gagttacttc 32521 ctatcaagcc agtaccgtgc taacaggctc aatattcctg aatgaaatat cagactagtg 32581 acaagctcct ggtcttgaga tgtcttctcg ttaaggagat gggccttttg gaggtaaagg 32641 ataaaatgaa tgagttctgt catgattcac tattctagaa cttgcatgac ctttactgtg 32701 ttagctcttt gaatgttctt gaaattttag actttctttg taaacaaatg atatgtcctt 32761 atcattgtat aaaagctgtt atgtgcaaca gtgtggagat tccttgtctg atttaataaa 32821 atacttaaa

By “agent” is meant a peptide, nucleic acid molecule, or small compound.

By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Methods of preparing antibodies are well known to those of ordinary skill in the science of immunology. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Tetramers may be naturally occurring or reconstructed from single chain antibodies or antibody fragments. Antibodies also include dimers that may be naturally occurring or constructed from single chain antibodies or antibody fragments. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab′) 2, as well as single chain antibodies (scFv), humanized antibodies, and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′) 2, and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies, such as camelid antibodies (Riechmann, 1999, Journal of Immunological Methods 231:25-38), composed of either a VL or a VH domain which exhibit sufficient affinity for the target, and multispecific antibodies formed from antibody fragments. The antibody fragment also includes a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody.

Antibodies can be made by any of the methods known in the art utilizing a polypeptide of the invention (e.g., an arenavirus GP1 polypeptide), or immunogenic fragments thereof, as an immunogen. One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The immunogen will facilitate presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in a number of ways. Nucleic acid sequences encoding a polypeptide of the invention or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in turn express the receptor on the cell surface generating an immunogenic response in the host. Alternatively, nucleic acid sequences encoding the polypeptide, or immunogenic fragments thereof, can be expressed in cells in vitro, followed by isolation of the polypeptide and administration of the polypeptide to a suitable host in which antibodies are raised.

Alternatively, antibodies against the polypeptide may, if desired, be derived from an antibody phage display library. A bacteriophage is capable of infecting and reproducing within bacteria, which can be engineered, when combined with human antibody genes, to display human antibody proteins. Phage display is the process by which the phage is made to ‘display’ the human antibody proteins on its surface. Genes from the human antibody gene libraries are inserted into a population of phage. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.

Antibodies made by any method known in the art can then be purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.

Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition (e.g., Pristane).

By “anti-GP1 antibody” is meant an antibody that selectively binds an arenavirus GP1 polypeptide, including for example the GP1 polypeptide of a New World arenavirus. In various embodiments, the anti-GP1 antibody specifically binds a GP1 receptor-binding site. Exemplary anti-GP1 antibodies include GD01, CR1-28, and CR1-07. In various embodiments, the anti-GP1 antibody has at least about 85% or greater amino acid identity to a CR1-28 or CR1-07 amino acid sequence provided below.

CR1-07 CDR H1 sequence GFTFGTSI CR1-07 CDR H2 sequence ISHDESRK CR1-07 CDR H3 sequence AKDLSPPYSYAWDIFQYW CR1-07 CDR L1 sequence QSVLYSSRSDNKY CR1-07 CDR L2 sequence WAS CR1-07 CDR L3 sequence QQYYSSPPTF CR1-28 VH sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSSAMHWVRQAPGKGLE WVAVIWSDGSNENYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYY CATDKTYVSGYTSTWYYFNYWGQGTLVTV S CR1-28 VL sequence DIQMTQSPSTLSASVGDRVTITCRASQSIDNWLAWYQQKPGKAPKLLIY TASRLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHRTFGQ GTKVEIK CR1-28 CDR H1 sequence GFTFSSA CR1-28 CDR H2 sequence IWSDGSNE CR1-28 CDR H3 sequence ATDKTYVSGYTSTWYYFNY CR1-28 CDR L1 sequence QSIDNW CR1-28 CDR L2 sequence KAS CR1-28 CDR L3 sequence QHRT CR1-07 VH sequence QVQLVESGGGVVHPGRSLRLSCAASGFTFGTSIMHWVRQAPGKGMQW VAQISHDESRKFYSDSVKGRFTVSRDNSKNTLFLEMSSLRIEDTAVYYCA KDLSPPYSYAWDIFQYWGQGSLVTVS CR1-07 VL sequence DIVMTQSPESLAVSLGERATINCKSSQSVLYSSRSDNKDYLAWYQQKP GQSPKLLIYWASTRESGVPERFTGSGSGTDFTLSISSLQAEDVAVYYC QQYYSSPPTFGGGTKVELK

In other embodiments, the anti-GP1 antibody has at least about 85% or greater amino acid identity to a GD01 amino acid sequence provided below.

GD01 CDR H1 sequence NYWMQ GD01 CDR H2 sequence AVYPGDGDTRFSQKFKG GD01 CDR H3 sequence ARRRVYYGSNYIYALDY GD01 CDR L1 sequence QNVGSA GD01 CDR L2 sequence SAS GD01 CDR L3 sequence QQYSSYPLAF CR1-07 VH sequence EVKLQQSGAELARPGTSVKLSCKASGYTFTNYWMQWIKQRPGQGLEWIG AVYPGDGDTRFSQKFKGKATLTADKSSSTAYMQLSSLSSEDSAVYFCAR RRVYYGSNYIYALDYWGQGTSVTVSAAKTTAPSVYPLAPVCGDTTGSSV TLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTS STWPSQSITCNVAHPASSTKVDKKIEPRVPKGEFQHTGGRY CR1-07 VL sequence DIVMTQSQKFMSTSIGDRVSITCKASQNVGSAVAWYQQKPGQSPKLLIY SASNRYTGVPDRFIGSESGTDFTLTISNMQSEDLADYFCQQYSSYPLAF GAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVK WKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA THKTSTSPIVKSFNRNEC

By “biological sample” is meant any liquid, cell, or tissue obtained from a subject. In some embodiments, the biological sample is blood.

In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In various embodiments, the disease is a viral hemorrhagic fever.

By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.

The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “marker” is meant any clinical indicator, protein, metabolite, or polynucleotide having an alteration associated with a disease or disorder.

As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a murine, bovine, equine, canine, ovine, or feline. In one aspect, the subject is a human.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, a therapeutic that “prevents” a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of a JUNV GP1-neutralizing antibody complex. Ribbon diagram of JUNV GP1 (left side of structure) bound to the GD01 Fab (heavy chain (V_(H), C_(H)1) shown below the light chain (V_(L), V_(H), V_(L), C_(H)1, and C_(L), respectively denote the antibody variable heavy, variable light, constant heavy 1, and constant light chain domains. The antibody CDR loops (CDR L1, CDR L3, CDR H1, CDR H2, and CDR H3) that contact GP1 are labeled. GP1 glycans are shown as sticks. GP1 disulfides are shown in yellow.

FIG. 2 depicts details about the JUNV GP1-GD01 interface. The GD01 variable heavy (V_(H)) and variable light (V_(L)) interacting segments are shown along with JUNV GP1 in ribbon diagram with a semi-transparent surface representation. The color scheme is as presented in FIG. 1. Top left: CDR H3 includes a cluster of three tyrosines that fit into a shallow groove on the concave face of GP1. Bottom left: CDRs H1 and H2 form a network of polar contacts with GP1 Loop 7 and the C-terminal end of loop 3. Top right: CDR L1 contacts the conserved GP1 Asn178 glycan. Bottom right: CDR L3 provides additional contacts to GP1 loop 3. The asterisk indicates a potential glycan for which density could not be observed.

FIGS. 3A-3C depict the overlap of GP1 receptor and GD01 footprints on GP1. FIG. 3A depicts a structural comparison of JUNV GP1 and MACV GP1. Left panel: Overlay of ribbon diagrams of JUNV GP1 and MACV GP1 (from PDB ID: 3KAS). N-linked glycans are shown as sticks (for MACV and JUNV), and disulfides are shown. Right panel: Superposition of JUNV GP1 onto the MACV GP1:TfR1 structure (PDB ID: 3KAS). MACV GP1 is omitted for clarity. FIG. 3B depicts a comparison of the footprint of GD01 and the predicted footprint of GP1 receptor TfR1 on the surface of JUNV GP1. Left panel: Surface representation of JUNV GP1 with the antibody V_(H) and V_(L) chain footprints colored in light and dark, respectively. An overlapping contact is shown as indicated. Right panel: Surface representation of JUNV GP1 with predicted TfR1 footprint shown. FIG. 3C depicts a comparison of GD01 CDRs and TfR1. Left panel: GD01 CDRs L1 and L3 and CDRs H1, H2 and H3 form two ridges (shown in red) that the antibody present to GP1. Tyr98 from the antibody heavy chain is shown as sticks. Right panel: TfR1 also presents two ridges to GP1 formed by the helix αII-2, the βII-2 strand, and loop βII-ββII-7a in its apical domain. Tyr211 in the βII-2 strand of the receptor is shown as sticks.

FIGS. 4A-4C show that the GP1 Tyr211-TfR1 pocket is an antibody target. FIG. 4A depicts a comparison of JUNV GP1 interaction with GD01 and MACV GP1 interaction with TfR1. Left panel: Ribbon diagram of JUNV GP1 with GD01 CDR H3 residues 97 to 100a shown as sticks. Residues labeled with an asterisk are mutated in the JUNV GP1_(mut) construct. The refined 2F_(o)-F_(c) electron density at 1 σ for antibody segment is shown. Right panel: Ribbon diagram of MACV GP1 with TfR1 βII-2 strand residues 209 to 212 shown as sticks (from PDB ID: 3KAS¹²). The refined 2F_(o)-F_(c) electron density at 1 σ for receptor segment is shown. FIG. 4B are a set of graphs depicting AHF survivor IgG binding to JUNV GP1 or JUNV GP1_(mut). ELISA of AHF survivor IgG binding to plates coated with JUNV GP1 or JUNV GP1_(mut). Lujo virus (LUJV) GP1 coated wells were included as a control. The pre-determined neutralization titer of each survivor is shown between parentheses. Error bars indicate standard deviation. FIG. 4C shows a set of graphs depicting competition of JUNV infection (Argentine Hemorrhagic Fever, AHF) survivor IgG with GD01 for binding of JUNV GP1. Competition ELISA: GD01 or 17b competitor IgG was added at increasing concentrations to plates coated with JUNV GP1, and the indicated AHF survivor IgG were added at fixed concentrations. Survivor IgG that bound to the plate was detected using a secondary anti-human HRP-conjugated antibody. Error bars indicate standard deviation.

FIGS. 5A-5E show that single B-cell sorting identified JUNV GP1-reactive antibodies. FIG. 5A is a schematic of an experiment to identify JUNV GP1-specific antibodies. PBMCs from CR1 were mixed with fluorescently labeled GP1, or GP1 and GP1_(mut) (Tyr211_(TfR1) pocket mutant). Right panel shows a FACS density plot for memory B cells that stained positive for GP1 (Sort 1), or positive for GP1, but negative for GP1_(mut) (Sort 2). The approximate location of the sorting gate is shown in dashed lines. CD19 is a B-cell marker. PE and PerCP are fluorophores. FIG. 5B are graphs showing ELISA data for the indicated identified monoclonal antibodies binding to JUNV GP1 or GP1_(mut). LUJV GP1 is a control. FIG. 5C are graphs showing kinetic analysis of binding for the Fabs of the indicated antibodies to immobilized JUNV GP1 as measured by SPR. CR1-07, CR1-10, and CR1-28 bind GP1 with affinities of 86.2 nM, 45 pM, 5.3 nM, respectively. Binding of monomeric Fabs for CR1-06 and CR1-09 to GP1 could not be detected (because of low affinity, data not shown). FIG. 5D depicts neutralization profiles of the indicated antibodies for GFP-expressing JUNV pseudotypes (entry levels measured by FACS). CR1-09 did not neutralize JUNV and CR1-10 poorly neutralized JUNV (data not shown). FIG. 5E depicts neutralization profiles of CR1-07 and CR1-28 IgG of JUNV and MACV pseudotypes. VSIV pseudotypes are a control.

FIGS. 6A-6D depict structures of GP1 complexes. FIG. 6A is a ribbon diagram of MACV GP1 (left in diagram) bound to TfR1 (right in diagram). PDB 3KAS. FIG. 6B is a ribbon diagram of JUNV GP1 (left in diagram) bound to the Fab of a murine neutralizing antibody (GD01). PDB 5EN2. FIG. 6C is a ribbon diagram of JUNV GP1 (middle of diagram) bound to the Fabs of novel fully human neutralizing antibodies CR1-28 (right of diagram) and CR1-10 (left of diagram). FIG. 6D is a ribbon diagram of MACV GP1 (top of diagram) bound to the Fab of novel cross-neutralizing human antibody CR1-07 that has activity against MACV and JUNV. All potent neutralizing antibodies studied thus far bind the receptor-binding surface of GP1. (GD01, CR1-28, CR1-07 or CR1-10).

FIG. 7 presents graphs depicting high affinity GD01 and QC03 binding to JUNV GP1. Biotinylated JUNV GP1 was immobilized on the surface of a streptavidin-coated sensor chip. GD01 (left panel) or QC03 (right panel) were passed at 100, 50, 25, 12.5, and 6.25 nM over the sensor chip with regeneration between steps for multi-cycle kinetic analysis. All injections were carried out in duplicate. The recorded sensograms (one of the duplicates) and the fitted curves, calculated using a 1:1 Langmuir binding model, are shown. The recorded sensograms essentially superimpose on the fitted curves. The estimated K_(D) for GD01 Fab and QC03 Fab for binding to JUNV GP1 are 12.5 and 1.5 nM, respectively. Binding constants are summarized in Table 1.

FIGS. 8A-8E depict GP1 sequences of New World hemorrhagic fever arenaviruses and design of receptor-binding site mutant. FIG. 8A is sequence alignment of JUNV GP1 residues 85-235 with the corresponding residues of the GP1 proteins of the New World mammarenaviruses MACV, TCRV, GTOV, SABV, and CHAPV. Empty circles indicate JUNV GP1 residues predicted to only contact TfR1, half-filled circles indicate JUNV GP1 residues only contacted by GD01, and filled circles indicate JUNV GP1 residues that are both predicted to contact TfR1 and interact with GD01. Tree diagrams indicate sites of N-linked glycosylation in JUNV GP1. Conserved cysteines and sites of N-linked glycosylation are highlighted in yellow and grey, respectively. The asterisk indicates the site of attachment of a conserved glycan contacted by GD01. FIG. 8B depicts a surface representation of JUNV GP1. The predicted TfR1 footprint is shown, and the Tyr211_(TfR1) pocket is circled. The sites of the substitutions introduced to generate the GP1_(mut) construct are shown in within the circle.

FIG. 8C is a size exclusion chromatography profile of JUNV GP1 (solid line) and JUNV GP1_(mut) (dashed lines). Both proteins elute at a similar retention volume when passed over a size exclusion column. The trace shown is for each protein after the nickel affinity purification step and removal of the His₆ tag. FIG. 8D are graphs showing ELISA binding data. ELISA binding of GD01 IgG to plates coated with JUNV GP1 or JUNV GP1_(mut) (Left panel). ELISA binding of QC03 IgG to plates coated with JUNV GP1 or JUNV GP1_(mut) (Right panel). LUJV GP1 is a control. Error bars indicate standard deviation. FIG. 8E is a graph depicting GD01 competition ELISA results. QC03 Fab or 17b competitor IgG were added at increasing concentrations to plates coated with JUNV GP1, and GD01 IgG was added at fixed concentrations. GD01 IgG that bound to the plate was detected using a secondary anti-mouse Fc HRP-conjugated antibody. Error bars indicate standard deviation.

FIGS. 9A-9D show that Survivor plasma contains GP1-directed antibodies, related to FIG. 4. FIG. 9A is a list of pre-determined neutralization titers for survivor plasma samples AHF1 through AHF10. PRNT₈₀=plaque neutralization reduction of 80%: N.T.=no titer. FIG. 9B are graphs showing data for HEK293T cells challenged with JUNV pseudotype after pre-incubation with purified IgG for the indicated survivor samples (Left panel). Entry levels were measured by FACS for GFP expression. ELISA data of the indicated survivor IgG samples with JUNV GP1 coated plates (Right panel). LUJV GP1 coated plates are included as a control. Error bars indicate standard deviation. FIG. 9C presents graphs showing HEK293T cells challenged with JUNV pseudotype after pre-incubation with purified IgG for the indicated survivor samples, with entry levels measured as in FIG. 9B. VSIV pseudotype virus is included as a control. FIG. 9D are graphs showing ELISA data of the indicated survivor IgG samples with JUNV GP1 coated plates. LUJV GP1 is included as a control. Error bars indicated standard deviation.

FIG. 10 is a bar graph depicting that GD01 does not neutralize the other New World hemorrhagic fever mammarenaviruses. GD01 was incubated at 100 μm/ml with JUNV, MACV, GTOV, SABV, CHAPV, Tacaribe virus (TCRV) pseudoviruses, or LASV or VSIV control pseudoviruses for 30 minutes. 293T cells were then challenged for 3 hr. Entry levels were measured by FACS for GFP expression 48 hr later, and normalized to levels in the absence of antibody (‘No Ab’, set at 100%). Error bars indicate standard deviation.

The atomic coordinates of the protein structure of a CR1-10/JUNV/CR1-28 complex are deposited in the Protein Data Bank (PDB) under Accession No. PDB ID 5W1K. The atomic coordinates of the protein structure of a MACV/CR1-07 complex are deposited in the Protein Data Bank (PDB) under Accession No. PDB ID 5WIM. The PDB file for JUNV GP1 bound to GD01 is available at Protein Data Bank Accession No. PDB ID 5EN2. The PDB file for MACV bound to transferrin is available at Protein Data Bank Accession No. PDB ID 3KAS. The atomic coordinates of the protein structure of an unliganded Fab fragment of CR1-07 is deposited at Protein Database (PDB) ID 5WIG. The entire contents of the protein structural data and atomic coordinates of these deposits are incorporated herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for treating or preventing arenavirus infection, as well as methods for the discovery or identification of therapeutic agents useful for inhibiting arenavirus infection. As described herein, the structure of the JUNV surface glycoprotein receptor-binding subunit (GP1) bound to a neutralizing monoclonal antibody was determined. The antibody engages the GP1 site that binds transferrin receptor 1 (TfR1)—the host cell surface receptor for all New World hemorrhagic fever arenaviruses—and mimics an important receptor contact. The invention is based, at least in part, on the discovery that the GP1 receptor-binding site (RBS) with which the New World hemorrhagic fever arenaviruses engage their obligate cell surface receptor, TfR1, is readily accessible to neutralizing antibodies. Several enveloped RNA viruses cause human viral hemorrhagic fevers, but passive immunotherapy has been rigorously shown to be effective in humans only for the treatment of JUNV infection. Without being bound by theory, it is proposed that RBS accessibility explains the effectiveness of convalescent-phase plasma therapy against JUNV. It is proposed that this functionally conserved epitope is a potential target for therapeutics and vaccines to limit infection by all New World hemorrhagic fever arenaviruses and also antibodies with cross-neutralizing activity against various viruses within the group. Thus, deploying and adapting this approach has the potential to limit outbreaks of the New World mammarenaviruses which depend on TfR1 for cellular entry, including the related arenaviruses MACV, GTOV, CHAPV, and SBAV.

Arenaviruses

Arenaviruses are enveloped viruses that carry single-stranded, bi-segmented RNA genomes. They include viruses found in captive alethinophidian snakes (the reptarenaviruses) and viruses that circulate mostly in rodents (the mammarenaviruses) (Radoshitzky et al., 2015). The arenaviruses are divided into two groups—‘Old World’ and ‘New World’—based on their serology and geographic distribution. They cause acute human viral hemorrhagic fevers with high case fatality rates (Paessler and Walker, 2013). The pathogenic Old World arenaviruses include Lassa (LASV) and Lujo (LUJV) viruses (Briese et al., 2009; Charrel and de Lamballerie, 2003). The New World arenaviruses include Junín (JUNV), Machupo (MACV), Guanarito (GTOV), and Sabiá (SBAV) viruses, which respectively cause Argentine (AHF), Bolivian, Venezuelan, and “Brazilian” hemorrhagic fever (Charrel and de Lamballerie, 2003; Oldstone, 2002; Salas et al., 1991). The most recently described member, Chapare virus (CHPV), was isolated from a small outbreak in Bolivia from 2003 to 2004 (Delgado et al., 2008). All cause severe human disease associated with hemorrhage and hemodynamic shock. Argentine hemorrhagic fever (AHF) is unique among viral hemorrhagic fevers because infusion of polyclonal neutralizing antibody-containing immune plasma derived from survivors (‘passive immunity’) is a well-established means of treating acute human infection (Maiztegui et al., 1979; Ruggiero et al., 1986). When provided within 8 days of illness, it decreases the case fatality rate from 15-30% to less than 1% (Maiztegui et al., 1979; Ruggiero et al., 1986). For it to be effective, the immune plasma has to be administered in defined doses of neutralizing activity (Enria et al., 1984). Without being bound by theory, this indicates that antibody-mediated virus neutralization is its main mode of action.

Arenavirus Surface Envelope Glycoprotein (GPC)

The arenavirus surface envelope glycoprotein (GPC) is the target of neutralizing antibodies. GPC comprises three non-covalently associated polypeptides; the stable signal peptide (SSP), GP1, and GP2 (Burri et al., 2012). GP1 binds cellular receptors, and GP2 contains a transmembrane segment and promotes fusion of the viral and host cell membranes. The ubiquitously expressed iron-uptake protein TfR1 is a cellular receptor for all New World hemorrhagic fever arenaviruses (Helguera et al., 2012; Radoshitzky et al., 2007). TfR1 orthologs from the natural hosts of all tested clade B New World arenaviruses are receptors for their corresponding virus, but only the New World arenaviruses that cause human disease bind human TfR1 (Choe et al., 2011).

Previously the structure of a MACV GP1-TfR1 complex was determined (Abraham et al., 2010). MACV GP1 binds TfR1 through an extensive network of contacts with the lateral surface of the apical domain of TfR1. Sequence comparison for the five New World hemorrhagic fever arenavirus GP1s show these to be complementary to the same TfR1 surface. A pocket on GP1 that accepts a tyrosine on the βII-2 strand of the TfR1 apical domain (Tyr211_(TfR1)) is a central feature of the GP1 receptor-binding site (RBS) (Abraham et al., 2010). This tyrosine is present on all the TfR1 orthologs that support entry of New World arenaviruses and is an important determinant of host specificity (Abraham et al., 2009; Radoshitzky et al., 2008).

Arenavirus Neutralizing Antibodies

GD01-AG02 (GD01) and QC03-BF11 (QC03) are antibodies that were generated in mice by immunization with inactivated JUNV (Sanchez et al., 1989). They belong to a small group of described monoclonal antibodies that neutralize JUNV, and they are active against infectious virus (Sanchez et al., 1989). However, their epitopes have not previously been characterized. As described herein, the X-ray crystal structure of JUNV GP1 complexed with the antigen-binding fragment (Fab) of GD01 was determined to understand how antibodies neutralize JUNV. The structure reveals that the antibody and receptor have similar modes of GP1 recognition and that the antibody's complementarity-determining region (CDR) H3 mimics the Tyr211_(TfR1) receptor contact. GD01 and QC03 compete for the same GP1 surface. Without being bound by theory, this indicates that both antibodies neutralize the virus by a similar mechanism. It is further shown that survivor immune plasma with neutralizing activity contains antibodies that target the Tyr211_(TfR1) pocket and GP1 RBS. The GP1 RBS is thus an accessible target for therapeutics and vaccines to limit infection caused by this important group of emerging human pathogens.

Therapeutic Methods

The methods and compositions provided herein can be used to treat or prevent an arenavirus infection. The methods and compositions provided herein can generate or enhance an immune response in a subject against an arenavirus infection. In general, arenavirus GP1 polypeptides and/or antibodies specific to arenavirus GP1 polypeptides described herein can be administered therapeutically and/or prophylactically to simulate an immune response specific for arenavirus GP1 antigen. The methods include administering an immunologically effective amount of an immunogenic GP1 polypeptide provided herein, and/or an immunologically effective amount of an antibody provided herein (e.g., CR1-07, CR1-28) to an individual in a physiologically acceptable carrier. In certain embodiments, the serum or plasma of an arenavirus immune survivor is used to treat or prevent the infection of another or different species of arenavirus.

The present invention provides methods of treating or preventing an arenavirus infection (e.g., a New World arenavirus infection), and/or disorders or symptoms thereof, which comprise administering a therapeutically effective amount of an anti-arenavirus GP1 agent as described herein (e.g., CR1-07, CR1-28, and/or a compound that specifically binds the TfR1 RBS of GP1), to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to an arenavirus infection, disease or symptom thereof (e.g., viral hemorrhagic fever). The method includes the step of administering to the mammal a therapeutic amount of an anti-GP1 agent (e.g., anti-GP1 antibody) sufficient to treat the infection, disease or symptom thereof, under conditions such that the infection, disease or disorder is treated.

The present invention also provides methods of treating or preventing an arenavirus infection (e.g., a New World arenavirus infection), and/or disorders or symptoms thereof, which comprise administering a therapeutically effective amount of an immunogenic composition or vaccine as described herein (e.g., comprising a polypeptide comprising the TfR1 RBS of GP1), to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of preventing an arenavirus infection in a subject susceptible to an arenavirus infection, disease or symptom thereof (e.g., viral hemorrhagic fever). The method includes the step of administering to the mammal a prophylactic amount of an immunogenic GP1 polypeptide (e.g., comprising the TfR1 RBS of GP1) sufficient to prevent the infection, disease or symptom thereof, under conditions such that the infection, disease or disorder is prevent.

Treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for an arenavirus infection, disease or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker (such as levels of S100 ligands), family history, and the like). The methods herein also include administering to the subject (including a subject identified as in need of such treatment) an effective amount of an anti-arenavirus GP1 antibody, an immunogenic composition or vaccine as described herein. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

In some aspects, the invention features methods of treating or preventing an arenavirus arenavirus infection or arenavirus-associated disease or condition (e.g., hemorrhagic fever) in a subject, the methods comprising administering to the subject an effective amount of a composition comprising an anti-arenavirus agent (e.g., an anti-GP1 antibody or therapeutic vaccine as described herein). Optionally, an anti-arenavirus therapeutic of the invention (e.g., an anti-GP1 antibody or therapeutic vaccine as described herein) may be administered in combination with one or more of any other standard anti-arenavirus therapies (see e.g., Vela et al., 2012). For example, an anti-GP1 antibody or therapeutic vaccine as described herein may be administered in combination with other antibodies or antibody cocktails with antiviral activity (including e.g., immune plasma), in combination with a vaccine (including e.g., a therapeutic vaccine), or in combination with a drug with anti-arenavirus activity (Ribavirin). Methods for administering combination therapies (e.g., concurrently or otherwise) are known to the skilled artisan and are described for example in Remington's Pharmaceutical Sciences by E. W. Martin.

Antibodies

As reported herein, antibodies that specifically bind arenavirus GP1 are useful in therapeutic methods. For example, antibodies that inhibit or target the binding of transferrin receptor 1 (TfR1) to glycoprotein 1 (GP1), are particularly useful in the methods of the invention. In particular embodiments, the invention provides methods of using anti-GP1 antibodies for the treatment or prevention of arenavirus infection and/or hemorrhagic disease. Exemplary anti-GP1 antibodies include one or more of GD01, CR1-28, and CR1-07, and antibodies obtained or isolated from survivors of arenavirus infection (e.g., from blood, serum, or plasma).

Methods of preparing antibodies are well known to those of ordinary skill in the science of immunology. As used herein, the term “antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term “antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab′)₂, and Fab. F(ab′)₂, and Fab fragments that lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983). The antibodies of the invention comprise whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab′, single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.

Unconventional antibodies include, but are not limited to, nanobodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062, 1995), single domain antibodies, single chain antibodies, and antibodies having multiple valencies (e.g., diabodies, tribodies, tetrabodies, and pentabodies). Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies have the affinity and specificity of conventional antibodies although they are only half of the size of a single chain Fv fragment. The consequence of this unique structure, combined with their extreme stability and a high degree of homology with human antibody frameworks, is that nanobodies can bind therapeutic targets not accessible to conventional antibodies. Recombinant antibody fragments with multiple valencies provide high binding avidity and unique targeting specificity to cancer cells. These multimeric scFvs (e.g., diabodies, tetrabodies) offer an improvement over the parent antibody since small molecules of ˜60-100 kDa in size provide faster blood clearance and rapid tissue uptake. See Power et al., (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy. Methods Mol Biol, 207, 335-50, 2003); and Wu et al. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting, 4, 47-58, 1999).

Various techniques for making and using unconventional antibodies have been described. Bispecific antibodies produced using leucine zippers are described by Kostelny et al. (J. Immunol. 148(5):1547-1553, 1992). Diabody technology is described by Hollinger et al. (Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993). Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) diners is described by Gruber et al. (J. Immunol. 152:5368, 1994). Trispecific antibodies are described by Tutt et al. (J. Immunol. 147:60, 1991). Single chain Fv polypeptide antibodies include a covalently linked VH::VL heterodimer which can be expressed from a nucleic acid including V_(H)- and V_(L)-encoding sequences either joined directly or joined by a peptide-encoding linker as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.

In various embodiments, an antibody that binds arenavirus GP1 is monoclonal. Alternatively, the anti-arenavirus GP1 antibody is a polyclonal antibody. In various embodiments, the antibody that binds arenavirus GP1 is obtained from the serum or plasma of an arenavirus immune survivor. The preparation and use of polyclonal antibodies are also known the skilled artisan. The invention also encompasses hybrid antibodies, in which one pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrids may also be formed using humanized heavy and light chains. Such antibodies are often referred to as “chimeric” antibodies.

In general, intact antibodies are said to contain “Fc” and “Fab” regions. The Fc regions are involved in complement activation and are not involved in antigen binding. An antibody from which the Fc′ region has been enzymatically cleaved, or which has been produced without the Fc′ region, designated an “F(ab′)₂” fragment, retains both of the antigen binding sites of the intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an “Fab”′ fragment, retains one of the antigen binding sites of the intact antibody. Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain, denoted “Fd.” The Fd fragments are the major determinants of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to immunogenic epitopes.

Antibodies can be made by any of the methods known in the art utilizing soluble polypeptides, or immunogenic fragments thereof, as an immunogen. One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The immunogen will facilitate presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in a number of ways. Nucleic acid sequences encoding human arenavirus GP1 or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in turn express the human arenavirus GP1 thereby generating an immunogenic response in the host. Alternatively, nucleic acid sequences encoding human arenavirus GP1 or immunogenic fragments thereof can be expressed in cells in vitro, followed by isolation of the human arenavirus GP1 and administration of the arenavirus GP1 to a suitable host in which antibodies are raised.

Alternatively, antibodies against arenavirus GP1 may, if desired, be derived from an antibody phage display library. A bacteriophage is capable of infecting and reproducing within bacteria, which can be engineered, when combined with human antibody genes, to display human antibody proteins. Phage display is the process by which the phage is made to ‘display’ the human antibody proteins on its surface. Genes from the human antibody gene libraries are inserted into a population of phage. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.

Antibodies made by any method known in the art can then be purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.

Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition (e.g., Pristane).

Monoclonal antibodies (Mabs) produced by methods of the invention can be “humanized” by methods known in the art. “Humanized” antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins. Techniques to humanize antibodies are particularly useful when non-human animal (e.g., murine) antibodies are generated. Examples of methods for humanizing a murine antibody are provided in U.S. Pat. Nos. 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.

Pharmaceutical Compositions

The present invention features compositions useful for treating or preventing arenavirus infection in a subject. The methods include administering an immunologically effective amount of a polypeptide provided herein, and/or an immunologically effective amount of an antibody provided herein to an individual in a physiologically acceptable carrier. In some embodiments, the composition comprises an anti-GP1 agent, such as an anti-GP1 antibody, or fragment thereof, as described herein. In other embodiments, the composition comprises an immunogenic GP1 polypeptide, such as a polypeptide comprising the TfR1 RBS of GP1, or fragment thereof, as described herein.

Typically, the carrier or excipient for the immunogenic composition or vaccine provided herein is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof. The preparation of such solutions ensuring sterility, pH, isotonicity, and stability is effected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, and the like. Such methods also include administering an adjuvant, such as an oil-in-water emulsion, a saponin, a cholesterol, a phospholipid, a CpG, a polysaccharide, variants thereof, and a combination thereof, with the composition of the invention. Optionally, a formulation for prophylactic administration also contains one or more adjuvants for enhancing the immune response to the GP1 polypeptide antigens. Suitable adjuvants include: complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and MF59.

The administration of a composition comprising an anti-arenavirus agent herein (e.g., anti-GP1) for the treatment or prevention of an arenavirus infection or arenavirus-associated disease or condition (e.g., hemorrhagic fever) may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing the disease symptoms in a subject. The composition may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, intraperitoneally, intramuscular, intrathecal, or intradermal injections that provide continuous, sustained levels of the agent in the patient. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the arenavirus infection or disease. Generally, amounts will be in the range of those used for other agents used in the treatment of cardiac dysfunction, although in certain instances lower amounts will be needed because of the increased specificity of the agent. A composition is administered at a dosage that ameliorates or decreases effects of the arenavirus infection or disease (e.g., hemorrhagic fever and symptoms thereof) as determined by a method known to one skilled in the art.

The therapeutic or prophylactic composition may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, intrathecally, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the heart; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a disease using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a cardiac dysfunction or disease, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) (e.g., an anti-GP1 agent described herein) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

In some embodiments, the composition comprising the active therapeutic (i.e., an anti-GP1 antibody herein) is formulated for intravenous delivery. As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

Methods of Identifying Agents that Inhibit Arenavirus GP1 Binding to Transferrin Receptor

In Silico Drug Design

The present invention permits the use of virtual design techniques (i.e., computer modeling or “in silico”) to design, select, and synthesize compounds capable of specifically binding arenavirus GP1, in particular, GP1-mediated cell attachment. In turn, these compounds may be effective in the treatment of an arenavirus infection or arenavirus-associated disease, such as hemorrhagic fever.

In addition to the more traditional sources of test compounds, computer modeling and searching technologies permit the rational selection of test compounds by utilizing structural information from the ligand binding sites and functional antibody binding sites (e.g., binding sites of arenavirus-inhibitory antibodies) on proteins of the present invention (e.g., arenavirus GP1). Such rational selection of compounds may decrease the number of compounds that may need to be screened to identify a therapeutic candidate compound. In various embodiments, the functional antibody binding site of GP1 is a TfR1 binding site. In some embodiments, the functional site on arenavirus GP1 comprises any one or more of amino acid residues 87-235 (JUNV numbering) of arenavirus GP1 or a corresponding region of an arenavirus GP1 (see, e.g., FIG. 8A). Important motifs include the pocket in the arenavirus GP1 that interacts with residue Tyr211 in transferrin receptor 1, which includes Junin virus GP1 residues Serine 111, Aspartate 113, Isoleucine 115, and Lysine 216, and the analogous residues in the GP1 proteins of MACV, GTOV, SBAV, CHPV, TCRV, and WWAV; and GP1 loop 3 (residues 113-124, JUNV numbering) and GP1 loop 7 (residues 166-174, JUNV numbering), and their respective counterparts in MACV, GTOV, SBAV, CHPV.

Knowledge of the protein sequences of the present invention may allow for generation of models of their binding sites that may be used to screen for potential agent(s) that bind to the binding sites. This process may be accomplished with the skills known in the art. One approach involves generating a sequence alignment of the protein sequence to a template (derived from the crystal structures or NMR-based model of a similar protein(s)), conversion of the amino acid structures and refining the model by molecular mechanics and visual examination. If a strong sequence alignment may not be obtained, then a model may also be generated by building models of the hydrophobic helices. Mutational data that point towards contact residues may also be used to position the helices relative to each other so that these contacts are achieved. During this process, docking of the known ligands into the binding site cavity within the helices may also be used to help position the helices by developing interactions that may stabilize the binding of the ligand. The model may be completed by refinement using molecular mechanics and loop building using standard homology modeling techniques. General information regarding modeling may be found in Schoneberg, T. et. al., Molecular and Cellular Endocrinology, 151:181-193 (1999), Flower, D., Biochim Biophys Acta, 1422, 207-234 (1999), and Sexton, P. M., Curr. Opin. Drug Discovery and Development, 2, 440-448 (1999).

Once the model is completed, it may be used in conjunction with one of several computer programs to narrow the number of compounds to be screened, e.g., the DOCK program (UCSF Molecular Design Institute, San Francisco, Calif. 94143) or FLEXX (Tripos Inc., MO). One may also screen databases of commercial and/or proprietary compounds for steric fit and rough electrostatic complementarity to the binding site. In one embodiment, the docking program is ZDOCK (Pierce et al., Bioinformatics. 2014 Jun. 15; 30(12):1771-3). In another embodiment, the docking program is AutoDock Vina (Trott et al., Journal of Computational Chemistry 31 (2010) 455-461).

In Silico Screening of Compounds

In one aspect, the invention provides means to carry out virtual screening of compounds using the disclosed atomic coordinates or coordinates derived therefrom. The atomic coordinates of the three-dimensional structure elucidated by the invention are input into a computer so that images of the structure and various parameters are shown on the display. The resultant data are input into a virtual compound library. Since a virtual compound library is contained in a virtual screening software, the above-described data may be input into such a software. Compounds may be searched for, using a three-dimensional structure database of virtual or non-virtual compounds, such as MDDR (Prous Science, Spain).

The potential interactions of a compound may be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques. If the theoretical structure of the given compound suggests insufficient interactions with arenavirus GP1, synthesis and testing of the compound may be obviated. However, if computer modeling indicates sufficient interactions, the molecule may then be synthesized and tested for its ability to regulate arenavirus GP1, using various methods described herein and/or that are known to a person skilled in the art.

Compounds may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to bind with individual binding sites or combinations thereof (e.g., P0, P+1, P−1) or other areas of arenavirus GP1.

One skilled in the art may use any of several methods to screen chemical entities or fragments for their ability to bind to arenavirus GP1 and more particularly with the specific binding sites or functional sites described herein (e.g., Protein Data Bank Accession No. PDB ID 5EN2, 3KAS, or those deposited under Protein Data Bank Accession No. PDB ID 5W1K and Protein Data Bank Accession No. PDB ID 5WIM. Sequences of arenavirus GP1, may also be threaded onto the protein backbone of an arenavirus GP1 crystal structure, with side chain positions optimized using methods known in the art. The resulting structural models may then be used to discover chemical entities or fragments that regulate arenavirus GP1 via in silico docking. The process may begin by visual inspection of, for example, the functional site on the computer screen based on the arenavirus GP1 coordinates presented in Protein Data Bank PDB ID 5EN2, 3KAS, or those deposited under Protein Data Bank Accession No. PDB ID 5W1K and Protein Data Bank Accession No. PDB ID 5WIM. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within a binding site of arenavirus GP1. Docking may be accomplished using software such as QUANTA™, SYBYL™ followed by energy minimization and molecular dynamics with molecular mechanics forcefields softwares, such as CHARMM™ and AIVIBER™.

Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include, but are not limited to, GRID™ (Goodford, P. J., J. Med. Chem., 28, 849-857 (1985)); MCSS™ (Miranker, A. and M. Karplus, “Proteins: Structure, Function and Genetics, 11, 29-34 (1991)); (3) AUTODOCK™ (Goodsell, D. S. and A. J. Olsen, Proteins: Structure, Function, and Genetics, 8, 195-202 (1990; DOCK™ (Kuntz, I. D. et al., J. Mol. Biol., 161, pp. 269-288 (1982)); GLIDE™ (Schrodinger Inc.); FLEXX™ (Tripos Inc); (7) GOLD™ (Jones et al., J. Mol. Biol., 245, 43-53, 1995).

Once suitable chemical entities or fragments have been selected, they may be assembled in silico or synthesized into a single compound. Chemical syntheses may be carried out by methods known in the art. In silico assembly may proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of arenavirus GP1. This may be followed by manual model building using softwares such as QUANTA™ or SYBYL™.

Useful programs for connecting the individual chemical entities or fragments include the following: CAVEAT™ (Bartlett, P. A. et al, Royal Chem. Soc., 78, 182-196 (1989)); 3D Database systems such as MACCS-3D™ (MDL Information Systems, San Leandro, Calif.); and HOOK™ (Molecular Simulations, Burlington, Mass.). In addition to building a compound in a step-wise fashion as described above, compounds may be designed as a whole or “de novo” using an empty active site or optionally including some portion(s) of a known compound. Such methods include, but are not limited to, LUDI™ (Bohm, H.-J., J. Com R. Aid. Molec. Design, 6, pp. 61-78 (1992)); LEGEND™ (Nishibata, Y. and A. Itai, Tetrahedron, 47, p. 8985 (1991)), and LEAPFROG™ (Tripos Inc., St. Louis, Mo.).

Once a compound has been designed or selected, the molecular interactions or affinity with which that compound may bind arenavirus GP1 may be tested and optimized by computational evaluation. For example, a compound may demonstrate a relatively small difference in energy between its bound and unbound states (i.e., a small deformation energy of binding). A compound may interact with arenavirus GP1 in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the unbound compound and the average energy of the conformations observed.

A compound that is designed or selected may be further computationally optimized so that in its bound state it may lack repulsive electrostatic interactions. Such interactions include repulsive charge-charge, dipole-dipole, and charge-dipole interactions. The sum of all electrostatic interactions between the compound and arenavirus GP1, may make a neutral or favorable contribution to the enthalpy of binding. Software programs to evaluate compound deformation energy and electrostatic interaction include, e.g., Gaussian 92™ (M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa.); AMBER™ (P. A. Kollman, University of California at San Francisco, Calif.); QUANTA/CHARMM™ (Molecular Simulations, Inc., Burlington, Mass.); and Insight II/Discover™ (Biosysm Technologies Inc., San Diego, Calif.).

Once a compound has been optimally selected or designed, substitutions may be made in some of its atoms or side groups in order to improve or modify its binding properties. Initial substitutions may be conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Such substituted compounds may then be analyzed for efficiency of fit to arenavirus GP1 by software programs similar to those described.

Crystallographic Evaluation of Chemical Entities for Binding to Arenavirus GP1

The invention allows one skilled in the art to study the binding of compounds to arenavirus GP1 by exposing either individual compounds or mixtures of compounds (such as may be obtained from combinatorial libraries) into arenavirus GP1 crystals or, alternatively, by co-crystallization of the compounds of interest with arenavirus GP1, using methods known in the art, or those described in the Examples herein. Acquisition and analysis of X-ray diffraction data from these crystals may then be performed using standard methods. If a compound binds to arenavirus GP1 then positive difference electron density will be observed in the Fourier maps calculated using the X-ray diffraction intensities and phases obtained from the arenavirus GP1 model presented herein. Models of the chemical entities may then be built into the electron density using standard methods, and the resulting structures may be refined against the X-ray diffraction data, providing experimental data describing the interaction of the compounds of interest. Those skilled in the art may use these models to design compounds based either on purely structural data; or on combination of structural data, biological/chemical activity based structure-activity relationship, and in silico drug design.

The compounds that are thus designed or selected may further be tested in an in vitro, in vivo, or ex vivo assays to determine if they bind or neutralize arenavirus GP1. Such assays are known to one skilled in the art, including functional assays such as ELISA, gel filtration, immunoprecipitation, plasmon resonance, and the like.

Kits

The invention provides kits for the treatment or prevention of an arenavirus infection. In some embodiments, the kit includes a therapeutic or prophylactic composition containing an effective amount of an anti-GP1 agent (e.g., an anti-GP1 antibody) in unit dosage form. In other embodiments, the kit includes a therapeutic or prophylactic composition containing an effective amount of an immunogenic agent (e.g., a GP1 polypeptide) in unit dosage form. In some embodiments, the kit comprises a device (e.g., nebulizer, metered-dose inhaler) for dispersal of the composition or a sterile container which contains a pharmaceutical composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired a pharmaceutical composition of the invention is provided together with instructions for administering the pharmaceutical composition to a subject having or at risk of contracting or developing an arenavirus infection. The instructions will generally include information about the use of the composition for the treatment or prevention of an arenavirus infection. In other embodiments, the instructions include at least one of the following: description of the therapeutic/prophylactic agent; dosage schedule and administration for treatment or prevention of arenavirus infection or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

Example 1. The Structure of the Complex of JUNV GP1 with GD01 was Determined

Because GP1 is expected to be the most membrane distal subunit of GP on the virion surface, GD01 and QC03 Fabs were tested for JUNV GP1-reactivity. QC03 and GD01 both bound JUNV GP1 with high affinity as measured by surface plasmon resonance (1.5 nM and 12.5 nM, respectively; FIG. 7 and Table 1).

TABLE 1 Binding rate constants of surface plasmon resonance analysis (FIG. 7). k_(a) k_(d) K_(D) Analyte Ligand (1/Ms) (1/s) (M) GD01 Fab JUNV GP1 2.08E5 2.61E−3 1.25E−8 QC03 Fab JUNV GP1 1.49E5 2.25E−4 1.51E−9 The structure of a GP1-neutralizing antibody complex was determined. A complex of JUNV GP1 with the GD01 Fab crystallized in space group P2₁2₁2₁. Molecular replacement with MACV GP1 (Abraham et al., 2010) and an unrelated Fab (Aoki et al., 2009) as search models was used and the structure was refined with data extending to 1.8 Å (FIG. 1, and Table 2).

TABLE 2 Data collection and refinement statistics (molecular replacement) (FIG. 1). JUNV GP1:GD01 Data collection P2₁2₁2₁ Cell dimensions a, b, c (Å) 52.1, 74.8, 177.6 Resolution (Å) 68.93 − 1.82 (1.92 − 1.82)* R_(merge) 0.226 (1.418) Mean //σ/ 6.7 (1.5) Completeness (%) 99.9 (99.9) Redundancy 5.9 (5.7) Refinement Resolution (Å) 68.93 − 1.82 No. reflections 63032 R_(work)/R_(free) 0.181/0.224 (0.239/0.274) No. atoms 5473 Protein 4462 Ligand/ion 125 Water 886 B-factors Protein 21 Ligand/ion 59 Water 38 R.m.s. deviations Bond lengths (Å) 0.010 Bond angles (°) 1.06 One crystal was used to collect the dataset. *Values in parentheses are for the highest-resolution shell.

The interface of JUNV GP1 with GD01 includes contacts from heavy-chain CDRs 1, 2 and 3 and light-chain CDRs 1 and 3, with the bulk of the interactions focusing on GP1 loops 3 and 7 (FIG. 1). The tyrosine-rich CDR H3 projects into a shallow cavity created by the curvature of the central β-sheet and loop 3 (FIG. 2, left upper panel), which is sandwiched between the heavy-chain and light-chain CDRs. CDRs H1 and H2 form a network of polar interactions with GP1 loops 3 and 7 (FIG. 2, left lower panel), and CDR L1 contacts the glycan attached at GP1 Asn178 (FIG. 2, right upper panel). This glycan is conserved in the GP1 proteins of all New World hemorrhagic fever mammarenaviruses. CDR L3 provides additional contacts to the N-terminal side of GP1 loop 3 (FIG. 2, right lower panel).

Example 2. Neutralizing Anti-GP1 Antibody and TfR1 Receptor have a Shared Mode of GP1 Recognition

JUNV GP1 is very similar to MACV GP1 (rmsd of 1.35 Å for Ca positions for residues 87-219 and 223-227), as expected from their sequences (48% identical for GP1 residues 87-235). However, there is a substantial difference in loop 10, in which MACV GP1 has a disulfide-linked insert with respect to JUNV (FIG. 3A, left panel). Because of this similarity, the JUNV GP1 and MACV GP1-TfR1 structures were superimposed to predict a TfR1 footprint on JUNV GP1 (FIG. 3A right panel and FIG. 3B). All but one of the 13 residues in that footprint are within the contact zone of the antibody, which includes a total of 15 residues (FIG. 8A).

When viewed from the perspective of GP1, the lateral surface of the TfR1 apical domain presents two parallel ridges—one formed by the edge of the αII-2 helix and the βII-6-βII-7a loop, and the other by the βII-2 strand (FIG. 3C). GP1 loop 3 fits between both ridges and crosses to the far side of the αII-2 helix. The antibody likewise presents two parallel ridges that accept GP1 loop 3—one from the heavy chain (parts of CDR H1, CDR H2, and CDR H3), and the other from the light chain (CDR L1 and CDR L3)—but closed off at one end by the C-terminal side of CDR H2. These two ridges superpose approximately onto the receptor βII-2 sheet and αII-2 helix ridges, respectively. The antibody thus resembles the receptor in the overall shape of its CDR surface.

Example 3. GD01 Tyr98 (Kabat) Fits into the Position Occupied in the GP1 Receptor-Glycoprotein Complex by TfR1 Tyr211

Close examination of the antibody-GP1 interface reveals that GD01 Tyr98 (Kabat numbering scheme) in CDR H3 fits precisely into the position occupied in the receptor-glycoprotein complex by Tyr211_(TfR1), even though most of the other specific interactions are different in character (FIGS. 4A and 4B). For example, the contact MACV GP1 Ser113 forms with the hydroxyl group of Tyr211_(TfR1) is replaced by a contact Asp113 in JUNV GP1 makes with the hydroxyl group of GD01 Tyr98. Another change at position 216, where lysine in JUNV GP1 replaces a threonine in MACV GP1, prevents the tyrosine side chain from reaching more deeply into the pocket. The orientation of Tyr98 in the GD01-GP1 complex probably is one that would be observed for Tyr211_(TfR1) in an authentic JUNV GP1-TfR1 complex, with CDR H3 of the antibody mimicking an important receptor contact.

A modified JUNV GP1 (designated JUNV GP1 . . . 0 was generated, in which the GP1 pocket that accepts Tyr211_(TfR1) was occluded by substituting residues that line it with bulkier ones (S111W, I115Y, and V117Y; FIG. 8B). JUNV GP1_(mut) expressed well in the supernatant of transfected HEK293T cells, and when purified eluted from a size exclusion column at the same retention volume as wild type GP1, indicating that this mutant protein is properly folded (FIG. 8C). As expected from the structure, GD01 did not bind JUNV GP1_(mut) (FIG. 8D, left panel). QC03, another JUNV neutralizing antibody, did bind JUNV GP1_(mut), but considerably more weakly than it did the WT protein (FIG. 8D, right panel), indicating that it likewise contacts the Tyr211_(TfR1) pocket, but probably less centrally than does GD01. QC03 Fab competed with GD01 for binding to JUNV GP1, confirming that both GD01 and QC03 are RBS-directed antibodies (FIG. 8E). Without being bound to theory, it is hypothesized that both neutralizing antibodies neutralize JUNV by interfering with binding to the host cell receptor.

Example 4. AHF Survivor Plasma Contained RBS-Directed Antibodies

Because survivor plasma transfusion is a very effective treatment for AHF (Enria et al., 1984; Maiztegui et al., 1979), it was determined whether immune plasma samples used for passive immunity contained RBS-directed antibodies. Nine survivor plasma samples (AHF1 through 9) were obtained with neutralizing antibody titers ranging from 1:10, 240 to 1:40, and a survivor plasma sample with no neutralizing activity at the time of collection (AHF10) was also obtained (FIGS. 9A-9D). Purified survivor IgG bound JUNV GP1, as measured by ELISA, with affinities that roughly correlated with their neutralizing activities (FIGS. 9A-9D).

Single B-cell sorting was used to identify JUNV GP1-reactive antibodies from the blood of a recipient of the live attenuated vaccine Candid #1 (the individual is referred to as Candid#1 Recipient 1, or CR1) (FIGS. 5A-5E). Five CR1 antibodies bound to JUNV GP1 by ELISA, and two antibodies, CR1-06 and CR1-28, bound to GP1_(mut) less tightly than WT GP1 (FIG. 5B). Without being bound by theory, this may indicate that the pocket is part of their epitope. CR1-28 was identified from a sort that included a counter-selection step with GP1_(mut) (Sort 1); the ELISA result therefore validates sensitivity of the platform to ‘fish out’ epitope specific antibodies. Fabs for three of the CR1 antibodies had high affinity for JUNV GP1 when measured by SPR (FIG. 5C), and three of the six antibodies neutralized JUNV pseudotypes (FIG. 5D). CR1-07 efficiently cross-neutralized MACV (FIG. 5E). CR1-28 at high concentrations (>100 μg/ml) also had activity against MACV.

Structures of GP1 complexes were determined for binding of the antibodies CR1-07, CR1-10, and CR1-28 isolated from single B-cell sorting to JUNV and MACV. The structure of MACV GP1 bound to TfR1 receptor (PDB 3KAS; see FIG. 6A) and the structure of JUNV GP1 bound to antibody GD01 (PDB 5EN2; see FIG. 6B) were determined. Structures of JUNV GP1 bound to CR1-28 and CR1-10 (FIG. 6C) and of CR1-07 bound to MACV GP1 (FIG. 6D) were determined. The JUNV GP1-CR1-28 co-crystal structure also included a poorly-neutralizing antibody (CR1-10) that does not bind the TfR1 receptor binding site in GP1. The structures revealed that CR1-28 neutralizes JUNV by targeting the Tyr211TfR1 pocket and also mimicking a contact made by Tyr211_(TfR1) (this antibody is therefore like GD01), and also binding GP1 loop 3 and GP1 loop 7. The structures also revealed that CR1-07 cross-neutralizes MACV by targeting a small, entirely conserved patch of the RBS that is remote from Tyr211_(TfR1) epitope but still includes GP1 loop 3 and loop 7. The structures thus define the GP1 TfR1 receptor binding site for potent neutralizing antibodies that can cross-neutralize different New World arenaviruses that bind the TfR1 receptor.

To determine if the Tyr211_(TfR1) pocket is a target for antibodies in human immune plasma, survivor IgG was tested for binding to JUNV GP1 and GP1_(mut). IgG purified from the plasma of AHF1 through AHF9 IgG bound JUNV GP1_(mut) more weakly than they bound WT GP1 (FIG. 4B). The difference was less marked for lower activity AHF8 and AHF9 IgG. Survivor IgGs (particularly those with high neutralizing activity) therefore contained antibodies that bind the GP1 Tyr211_(TfR1) pocket.

Although the Tyr211_(TfR1) pocket is a central feature of the GP1 RBS, it is only a small part of the predicted TfR1 footprint (FIG. 8B). Some antibodies that recognize nearby sites in the large RBS, but not the pocket itself, may also have neutralizing activity. An example of one such antibody is CR1-07 (FIG. 6D). Because the GD01 footprint encompasses the entire GP1 RBS, the most potent survivor IgGs were tested for reactivity against the GD01 epitope using a competition ELISA. IgG isolated from the plasma of AHF1 through 5, but not a control antibody (17b), competed with GD01 for binding to JUNV GP1 (FIG. 4C). These data confirmed that the RBS epitope is a target for antibodies generated during natural human infection.

The lack of complete competition of GD01 with survivor IgGs in the ELISA shown in FIG. 4C indicates that antibodies binding epitopes other than the GP1 RBS are present in survivor plasma. While antibodies that target the RBS with reasonable affinity should, in principle, be neutralizing, non-neutralizing antibodies may bind other epitopes in GP1. Potential non-neutralizing epitopes include GP1 surfaces involved in oligomerization that are accessible on soluble GP1, but not accessible on functional, trimeric GPC on the virion surface. These non-neutralizing antibodies could have been generated against shed JUNV GP1; GP1 shedding has been described in acute infection by another arenavirus, LASV (Branco et al., 2010). Without being bound by theory, CR1-10, which binds the non-receptor binding face of GP1 (FIG. 6C), may be one such antibody.

Receptor mimicry is a recurring phenomenon in antibody neutralization of enveloped RNA viruses. Receptor-mimicking antibodies neutralize influenza viruses (Schmidt et al., 2015; Xu et al., 2013) and HIV-1 (Scheid et al., 2011; Zhou et al., 2010). The results here reinforce the concept that host receptor mimicry is a general mode of antibody neutralization for diverse families of viruses.

GD01 does not neutralize the other New World hemorrhagic fever arenaviruses (FIG. 10). In contrast, CR1-28 and CR1-07 have activity against MACV. Sequence differences in the GP1 RBS probably block binding to GD01 but preserve its interaction with TfR1, CR1-28, and CR1-07. These differences result in part from long-term co-adaptation of viruses with their natural rodent hosts, including an “arms race” between the various rodent TfR1 orthologs and the mammarenavirus GP1s (Demogines et al., 2013). The lateral edge and tip of the TfR1 apical domain (FIGS. 3A and 3C, right panels), a site engaged by all New World mammarenaviruses, is a “hot spot” for mutations with strong evidence of selective pressure in rodents (Demogines et al., 2013). Without being bound by theory, a large RBS may allow GP1 to tolerate variation in host receptor sequences in a virus-host arms race, but also leaves it exposed for immune recognition. Neutralizing antibodies targeting this site could then more readily select for viral escape mutations, and thus account for RBS diversity as New World mammarenaviruses circulate in their respective rodent hosts.

Because GD01 and TfR1 recognize GP1 similarly, the structure could serve as a template for in vitro or in silico design of antibodies that more faithfully mimic the receptor and neutralize some or all of the other viruses in this group. For example, the relatively prominent CDR H3 (17 residues) of GD01 projects substantially farther from the contact surface with GP1 than does the βII-2 strand of TfR1, and residues at its tip would collide with the MACV GP1-specific loop 10 insert (FIG. 4B). An engineered antibody with a similar contact surface but a shorter CDR H3 might in principle neutralize both JUNV and MACV. Interestingly, both CR1-28 and CR1-07 avoid MACV GP1 loop 10. Without being bound by theory, this in part explains their ability to cross-react with MACV.

A less accessible RBS might explain why treatment with convalescent phase survivor plasma may be less effective against other viral hemorrhagic fevers. In the GP of the filoviruses Ebola virus (EBOV) and Sudan virus, for example, the GP1 RBS is hidden beneath a heavily glycosylated mucin-like domain that contains both O-linked and N-linked carbohydrates and becomes exposed only after this domain has been cleaved by cathepsin in acidified endosomes (Chandran et al., 2005; Lee et al., 2008). Neutralizing antibodies that target other sites, such as the GP1-GP2 interface (which lies near the viral membrane), appear to have a larger role in limiting these infections (Dias et al., 2011; Murin et al., 2014). The GP1 RBS for another filovirus that causes human hemorrhagic fevers, Marburg virus (MARV), is more exposed, and antibodies binding this site may be more important in controlling infection by this virus (Flyak et al., 2015). A MARV neutralizing antibody that probably mimics a viral glycoprotein-receptor contact has been described (Flyak et al., 2015; Hashiguchi et al., 2015).

Like filoviruses, arenaviruses that are endemic to South America all lack adequate and rapidly scalable treatment options. Antibodies like GD01, CR1-28, and CR1-07 could eventually replace immune plasma in the treatment of AHF and perhaps of other New World hemorrhagic fevers. The findings described herein further indicate that a recombinant GP1 subunit-based immunization strategy, which focuses the immune response on the RBS by hiding other sites, has the potential to effectively protect against infection caused by these lethal agents.

The results described herein were obtained using the following materials and methods.

Cells and Plasmids

HEK293T (human embryonic kidney cells, ATCC CRL-1268) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). GnTI^(−/−) 293S cells were maintained in serum free medium (FreeStyle™ 293 Expression Medium, Life Technologies). GP-expressor plasmids for JUNV, MACV, GTOV, SABV, CHAPV, TCRV, and LASV, and an expressor plasmid for vesicular stomatitis virus (VSIV) G, have been previously described (Abraham et al., 2009; Radoshitzky et al., 2007) (Helguera et al., 2012). LUJV GP (GenBank: NC_023776.1) was synthesized as a codon-optimized gene for mammalian expression, and subcloned into the pCAGGS vector. Hybridomas producing monoclonal antibodies GD01 and QC03 (clones GD01-AG02 and QC03-BF11, respectively) (Sanchez et al., 1989) were obtained from the NIAID Biodefense and Emerging Infections (BEI) repository. These cells in Hybridoma-SFM expression medium (Life Technologies). The pHLSec vector (Aricescu et al., 2006) was used to express secreted glycoproteins.

Pseudotype Transduction

Pseudotypes were packaged in 293T cells by transfecting plasmids encoding murine leukemia virus gag/pol, the arenaviral GP, and the pQCXIX transduction vector (BD Biosciences) expressing eGFP in a 1:1:1 ratio, as previously described (Radoshitzky et al., 2007). Virus-containing culture supernatant was harvested 24 hr and 48 hr later. Supernatants were filtered through a 0.45 μm membrane, and pseudotypes were stored at −80° C. until later use. For antibody neutralization experiments, pseudotypes were pre-incubated with polyclonal IgG or monoclonal antibodies for 30 min at 37° C. The pseudotypes and antibody mixture were then added to cells, and the media changed with 10% (v/v) FBS-supplemented DMEM 3 hr post transduction. Entry levels were measured by flow cytometry 48 hr post transduction.

Protein Expression and Purification

To generate biotinylated proteins, JUNV GP1 (residues 87-235), MACV GP1 (residues 87-250), and LUJV GP1 (residues 59-217), were each subcloned along with an N-terminal His₆-tag, followed by a Tobacco Etch Virus (TEV) protease site, a BirA ligase site (amino acids: GLNDIFEAQKIEWHE), and a seven residue linker (amino acid sequence: GTGSGTG), into the pHLSEC expression vector (Aricescu et al., 2006). JUNV GP1_(mut), which encodes JUNV GP1 residues 87-235 and contains the S111W, I115Y and V117Y mutations was generated by site directed mutagenesis. Proteins were produced by transfection using linear polyethylenimine in HEK293T cells grown in suspension, and the proteins purified using nickel affinity chromatography. The His₆-tag was removed with TEV protease and reverse nickel-affinity purification, and then site specific biotinylation was performed with BirA ligase followed by size-exclusion chromatography on a Superdex 200 column (GE Healthcare Sciences) to remove free biotin. For crystallography, JUNV GP1 (residues 87-235) was subcloned with the addition of N-terminal His₆-Tag, a TEV protease site, and a short linker (amino acids: SGSG), into the pHLSEC vector. The protein was produced in GnTI^(−/−) 293 S cells grown in suspension and purified by nickel affinity chromatography, and the tag was removed with TEV digestion, reverse nickel-affinity purification, and size exclusion on a Superdex 200 column. GD01 and QC03 Fabs were produced using a Pierce™ Fab Preparation Kit (Thermo scientific) from Protein G Ultralink® Resin (Thermo scientific) following the manufacturer's protocol.

Surface Plasmon Resonance Binding Assays

Binding experiments were performed in duplicate with a Biacore 3000 (GE Health Care Sciences), using streptavidin coated sensor chips, and biotinylated JUNV GP1. Approximately 600-800 response units of biotinylated JUNV GP1 were captured onto the chips to avoid rebinding events. Experiments were carried out in HBS-EP (10 mM HEPES pH 7.5, 150 mM NaCl, 3 mM EDTA, and 0.005% P-20). GD01, QC03, CR1-07, CR1-10, and CR1-28 Fabs were passed over the surface at various concentrations, and GP1-Fab interactions were analyzed using multi-cycle kinetic analysis with 2 min association and 5 min dissociation phases with a flow rate of 50 μL/min. Between each cycle, the surface was regenerated with two 5 μl injections of 35 mM NaOH, 1.3 M NaCl at 100 μL/min, and 2 minute stabilization after regeneration. For analysis, injections over blank surfaces were subtracted from the data, and the data was fit using a 1:1 Langmuir binding model in the BiaEvaluation software (GE Health Care Sciences).

Data Collection and Structure Determination

The JUNV GP1-GD01 Fab complex crystallized in the P2₁2₁2₁ space group. X-ray diffraction data were collected at wavelength of 0.9789 and temperature of 100° K at NE-CAT beam line ID-24C at the Advanced Photon Source (Argonne National Laboratory). Data were processed using MOSFLM (Leslie and Powell, 2007), and the structure of the complex was determined by molecular replacement with PHASER (McCoy et al., 2007) with MACV GP1 (PBD ID: 3KAS) (Abraham et al., 2010) and the 4F8 Fab (PDB ID: 3FMG) (Aoki et al., 2009) as search models. Electron density was observed for residues 87-227 for JUNV GP1, residues 1 to 213 with the exception of residues 128-132 in the GD01 HC, and residues 1 to 212 in the GD01 LC. We could form a ternary complex of JUNV GP1 bound to the Fabs of CR1-28 and CR1-10. This complex yielded crystals in space group P12₁1 that diffracted to 3.99 Å. We determined its structure using molecular replacement, and identified four copies of the complex in the asymmetric unit (ASU). We obtained crystals of a complex of a MACV GP1-CR1-07 in space group P4₂2₁2 that diffracted to 3.9 Å. Initial molecular replacement searches with MACV GP1 (PDB 3KAS). We determined the structure of the unliganded CR1-07 Fab, and used these coordinates along with MACV GP1 as search models to solve the structure of the complex. The model includes 4 copies of the complex per ASU.

Sequence for the GD01 Fab was obtained using a previously described protocol for antibody gene recovery from the parent hybridoma (Fields et al., 2013), and performed iterative model building with COOT (Emsley et al., 2010) and refinement with PHENIX (Adams et al., 2010), yielding a final R_(work) of 18.1% and R_(free) of 22.4% (Table S2), with Ramachandran favored: 97.7% and Ramachandran outliers: 0.18%. The JUNV GP1 CR1-28/CR1-10 structure has R_(work) 22.4% and R_(free) of 28.1%, and the MACV GP1 CR1-07 structure has R_(work) of 26.3% and R_(free) of 27.8%. Figures were made with the PyMol Molecular Graphics System, Schrodinger, LLC.

Human Immunoglobulin Purification

Five (5) de-identified plasma samples from Argentine hemorrhagic fever survivors were obtained from the immune plasma bank at the Instituto Nacional de Enfermedades Virales Humanas (INEVH), based in Pergamino, Argentina, where these samples are routinely stored. Provision of the previously collected survivor plasma samples was approved by the INEVH Ethics Committee, and the Harvard University Faculty of Medicine Committee on Human Studies (identified as not involving human subjects under 45CFR46.102(f)). An additional 5 plasma survivor samples were obtained through INEVH under a Boston Children's Hospital Institutional Review Board and INEVH Ethics Committee approved protocol (IRB: IRB-P00007578) after informed consent was obtained from all subjects. Neutralizing antibody titers from the donors at the time of plasma collection had previously been determined by the fixed-virus/variable serum technique, with Vero cell monolayers infected with the XJC1₃ attenuated strain of JUNV virus, and defined as a plaque neutralization reduction of 80% (PRNT₈₀). Because the heparin that is contained in the plasma samples could interfere with the interpretation of the results of the pseudotype assay, IgG from these samples were purified using Protein G Ultralink® Resin (Thermo scientific), according to manufacturer's instructions.

ELISA Experiments

Streptavidin-coated ELISA plates (Thermo scientific) were used. Wells were coated with biotinylated antigens at concentration of 0.2 μg/ml in PBS containing 2% bovine serum albumin. For ELISA-based competition assays, GD01 or 17b IgG were added at increasing concentrations during a pre-incubation step of 30 minutes, then the AHF survivor IgG was added at fixed concentrations (to obtain a baseline signal of 1.5-2 OD 450 nm). Bound antibody was detected with horse-radish peroxidase (HRP)-coupled anti-human antibody.

Single B Cell Sorting

Written informed consent was obtained from a healthy participant previously immunized with Candid #1 more than 2 years prior to study enrollment. This study was approved by the Boston Children's Hospital Institutional Review Board (IRB). Antigen-tetramers were prepared, and peripheral blood mononuclear cells were stained and washed as previously described (Franz et al., 2011), with the exception that phycoerythrein (PE)-labeled JUNV GP1 (Sort 1) and PE-labeled JUNV GP1 and PerCP-labeled JUNV GP1_(mut) (Sort 2) were used for tetramer preparation and cell staining. The mRNA pre-amplification, RT-PCR, and nested PCR steps were carried out as previously described (Franz et al., 2011), with the exception that oligo-dT primers were used in the RT-PCR step.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

The following references are cited herein:

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1. An isolated antibody or antigen-binding fragment thereof that specifically binds arenavirus glycoprotein 1 (GP1), the antibody comprising one or more complementary determining regions (CDR) selected from CDR H1 sequence GFTFGTSI CDR H2 sequence ISHDESRK CDR H3 sequence AKDLSPPYSYAWDIFQYW CDR L1 sequence QSVLYSSRSDNKY CDR L2 sequence WAS CDR L3 sequence QQYYSSPPTF CDR H1 sequence GFTFSSA CDR H2 sequence IWSDGSNE CDR H3 sequence ATDKTYVSGYTSTWYYFNY CDR L1 sequence QSIDNW CDR L2 sequence KAS; and CDR L3 sequence QHRT.


2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises the heavy chain sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSSAMHWVRQAPGKGLE WVAVIWSDGSNENYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYY CATDKTYVSGYTSTWYYFNYWGQGTLVTVS

and the light chain sequence DIQMTQSPSTLSASVGDRVTITCRASQSIDNWLAWYQQKPGKAPKLLIY TASRLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHRTFGQG TKVEIK

or an antibody comprises the heavy chain sequence QVQLVESGGGVVHPGRSLRLSCAASGFTFGTSIMHWVRQAPGKGM QWVAQISHDESRKFYSDSVKGRFTVSRDNSKNTLFLEMSSLRIEDTA VYYCAKDLSPPYSYAWDIFQYWGQGSLVTVS

and the light chain sequence DIVMTQSPESLAVSLGERATINCKSSQSVLYSSRSDNKDYLAWYQQK PGQSPKLLIYWASTRESGVPERFTGSGSGTDFTLSISSLQAEDVAVY YCQQYYSSPPTFGGGTKVELK.


3. The isolated antibody or antigen-binding fragment thereof of claim 1, that inhibits binding of GP1 and a transferrin receptor 1 (TfR1).
 4. The isolated antibody or antigen-binding fragment thereof of claim 1, that binds a TfR1 receptor binding site of GP1.
 5. The isolated antibody or antigen-binding fragment thereof of any one of claim 1, wherein the TfR1 receptor binding site comprises amino acids 87-235 of JUNV GP1 or corresponding amino acids of an arenavirus GP1.
 6. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the TfR1 receptor binding site comprises one or more of amino acids Serine 111, Aspartate 113, Isoleucine 115, and Lysine 216, amino acids 113-124 (JUNV GP1 loop 3), and amino acids 166-174 (JUNV GP1 loop 3) of JUNV GP1 or corresponding amino acids of an arenavirus GP1 7-13. (canceled)
 14. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the subject is infected or at risk of infection with a New World arenavirus.
 15. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the New World arenavirus is Junín (JUNV), Machupo (MACV), Guanarito (GTOV), Sabiá (SBAV), Chapare virus (CHPV), Tacaribe virus (TCRV), or White Water Arroyo virus (WWAV). 16-17. (canceled)
 18. An isolated polynucleotide encoding one or more sequences of the isolated antibody or antigen-binding fragment thereof of claim
 1. 19. An isolated vector comprising the polynucleotide of claim
 18. 20. An isolated cell comprising the vector of claim
 19. 21. (canceled)
 22. A method of inhibiting or preventing binding of a transferrin receptor 1 (TfR1) and a glycoprotein 1 (GP1) of one species of New World arenavirus, the method comprising contacting a TfR1 with an antibody or antigen-binding fragment thereof generated by an immune response to a glycoprotein 1 (GP1) of a second species of New World arenavirus. 23-24. (canceled)
 25. A method of treating or preventing a New World arenavirus infection, the method comprising administering to a subject infected or at risk of infection with one species of New World arenavirus an antibody or antigen-binding fragment thereof generated by an immune response to a second species of New World arenavirus. 26-39. (canceled)
 40. A method of inhibiting or preventing binding of a transferrin receptor 1 (TfR1) and an arenavirus glycoprotein 1 (GP1), the method comprising contacting a TfR1 with the isolated antibody or antigen-binding fragment thereof of claim
 1. 41-42. (canceled)
 43. A method of treating or preventing a New World arenavirus infection, the method comprising administering to a subject in need thereof the isolated antibody or antigen-binding fragment thereof of claim
 1. 44-52. (canceled)
 53. An immunogenic composition or vaccine comprising a polypeptide comprising a TfR1 binding site of a New World arenavirus GP1, wherein the TfR1 binding site comprises amino acids 87-235 of JUNV GP1 or corresponding amino acids of an arenavirus GP1. 54-58. (canceled)
 59. A method of generating an immune response in a subject, the method comprising administering to the subject the immunogenic composition of or vaccine of claim
 53. 60. An in silico method for identifying an agent that inhibits binding of a New World arenavirus GP1 to a transferrin receptor, the method comprising: a) generating a three-dimensional representation of a transferrin receptor structural binding pocket using the atomic coordinates of a New World arenavirus surface envelope glycoprotein amino acid residues in the sequence; and b) employing the three-dimensional structure to design or select an agent that inhibits binding.
 61. (canceled)
 62. A kit comprising the antibody or antigen-binding fragment thereof of claim
 1. 